MicroRNA (miRNA) and Downstream Targets for Diagnostic and Therapeutic Purposes

ABSTRACT

In some embodiments, the invention is directed to a method for diagnosing fibrosis and/or fibrosis related diseases and to a method for screening a pharmaceutically active compound for the treatment of fibrosis and/or fibrosis related diseases. The present invention further relates to compositions for use in the treatment, amelioration, and/or prevention of fibrosis. In certain embodiments, the compositions modulate the activity of a miRNA for the treatment, amelioration, and/or prevention of fibrosis. In certain embodiments, the compositions inhibit the activity of miR-21 for the treatment, amelioration, and/or prevention of fibrosis.

FIELD OF THE INVENTION

The present invention relates to the field of microRNA (miRNA), inparticular miR-21 and its down-stream targets for the diagnosis,prevention and/or therapy of fibrosis and other diseases. The presentinvention further relates to compositions, methods and uses for thetreatment for fibrosis. Such methods comprise modulating and inhibitingthe activity of a miRNA in a subject having fibrosis.

BACKGROUND OF THE INVENTION

MicroRNAs are a broad class of small non-coding RNAs that controldiverse biological processes including major signaling pathways byregulating the expression of complementary target mRNAs (Ambros, 2004).Dysregulation of microRNAs in various disease entities is caused byalterations in the genome (Mi et al., 2007), differential expression orviral infections in some cases changing microRNA function into tumorsuppressors or oncogenes. MicroRNAs were recently implicated in theregulation of diverse cardiac functions in a series of elegant geneticstudies (Care et al., 2007; Yang et al., 2007). Although these studieshelp to delineate the roles of microRNA in heart physiology, growth andmorphogenesis, detailed molecular mechanisms for microRNAs in diseasepathways in vivo are poorly understood. Single-stranded oligonucleotidemicroRNA antagonists have been shown to silence endogenous microRNAs invitro and in vivo with resulting effects on target mRNA and proteinlevels and metabolism (Kruetzfeldt et al., 2005; Esau et al., 2006).These findings pointed to the application of microRNA antagonists for invivo validation of microRNA function and, perhaps more importantly, as anovel therapeutic modality.

SUMMARY

The present invention relates to a promoter region of a microRNA, theuse of a microRNA, in particular miR-21, and related elements for thediagnosis and for the manufacture of a medicament for the treatmentand/or prevention of fibrosis and/or fibrosis related diseases.Additionally, the invention concerns various antisense oligonucleotidesagainst targets of miR-21. A cell deficient for miR-21, the promoterregion and targets of miR-21 and a knock-out organism thereof are alsoencompassed. Finally, the invention is directed to a method fordiagnosing fibrosis and/or fibrosis related diseases and to a method forscreening a pharmaceutically active compound for the treatment offibrosis and/or fibrosis related diseases.

Provided herein are methods for treating fibrosis, comprisingadministering to a subject having or suspected of having fibrosis acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence complementary to anmiRNA.

Provided herein are methods comprising identifying a subject having orsuspected of having fibrosis; and administering to the subject acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary a miRNA or a precursor thereof.

Provided herein are methods comprising administering to a subject atrisk for developing fibrosis a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence which complementary to a miRNA or a precursorthereof, thereby preventing fibrosis. In certain embodiments, thefibrosis is liver fibrosis. In certain embodiments, the fibrosis is lungfibrosis. In certain embodiments, the fibrosis is skin fibrosis. Incertain embodiments, the fibrosis is age-related fibrosis. In certainembodiments, the fibrosis is cardiac fibrosis. In certain embodiments,the fibrosis is kidney fibrosis. In certain embodiments, the fibrosis isspleen fibrosis.

Provided herein are methods comprising administering to a subject havingor suspected of having fibrosis a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence which is complementary to a miRNA or a precursorthereof, wherein the subject has at least one cardiac disease orcondition.

Provided herein are methods comprising identifying a subject having orsuspected of having fibrosis, wherein the subject has at least onecardiac disease or condition; and administering to the subject acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a miRNA or a precursor thereof.

In certain embodiments, the cardiac disease or condition is selectedfrom the group consisting of cardiac hypertrophy, hypertensive heartfailure, diastolic heart failure, systolic heart failure, heart-relatedstorage disease, cardiomyopathy, constrictive pericarditis, coronaryartery disease, acute myocardial infarction, chronic myocardialinfarction, right heart failure, cardiac arrhythmias,myocarditis-related fibrosis, and heart valve disease.

In certain embodiments the cardiomyopathy is selected from the groupconsisting of dilatative cardiomyopathy, hypertrophic cardiomyopathywith obstruction, hypertrophic cardiomyopathy without obstruction,restrictive cardiomyopathy, arrhythmogenic right ventricularcardiomyopathy, and diabetic cardiomyopathy.

In certain embodiments the heart valve disease is selected from thegroup consisting of mitral valve stenosis, aortic valve stenosis,tricuspidal valve stenosis, and pulmonary valve stenosis.

In certain embodiments the heart valve disease is selected from thegroup consisting of mitral valve insufficiency, aortic valveinsufficiency, tricuspidal valve insufficiency, and pulmonary valveinsufficiency.

In certain embodiments the methods provided herein further compriseadministering one or more additional pharmaceutical agents.

In certain embodiments the administering ameliorates heart weightincrease, left ventricular dilation, or impairment of fractionalshortening.

In certain embodiments the administering prevents heart weight increase,left ventricular dilation, or impairment of fractional shortening.

In certain embodiments the administering improves cardiac function.

In certain embodiments the administering comprises intravenousadministration, subcutaneous administration, intraarterialadministration, or intracardial administration.

Provided herein are methods for treating fibrosis, comprisingadministering to a subject having or suspected of having fibrosis acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence complementary to amiRNA or a precursor thereof, wherein the subject has at least one liverdisease or condition.

Provided herein are methods comprising identifying a subject having orsuspected of having fibrosis, wherein the subject has at least one liverdisease or condition; and administering to the subject a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a miRNA or a precursor thereof. Incertain embodiments, the at least one liver disease or condition ischronic liver injury. In certain embodiments, the at least one liverdisease or condition is hepatitis virus infection. In certainembodiments the hepatitis infection is hepatitis C virus infection. Incertain embodiments the at least one liver disease or condition isnon-alcoholic steatohepatitis. In certain embodiments the administrationcomprises intravenous administration or subcutaneous administration. Incertain embodiments the at least one liver disease or condition iscirrhosis. In certain embodiments the administering improves liverfunction.

Provided herein are methods for treating fibrosis, comprisingadministering to a subject having or suspected of having fibrosis acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence complementary to amiRNA or a precursor thereof, wherein the subject has at least one lungdisease or condition.

Provided herein are methods comprising identifying a subject having orsuspected of having fibrosis, wherein the subject has at least one lungdisease or condition; and administering to the subject a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a miRNA or a precursor thereof. Incertain embodiments the at least one other lung disease or condition ischronic obstructive lung disease. In certain embodiments theadministering comprises pulmonary administration.

Provided herein are methods for treating fibrosis, comprisingadministering to a subject having or suspected of having fibrosis acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence complementary to amiRNA or a precursor thereof, wherein the subject has at least one otherdisease or condition.

Provided herein are methods comprising identifying a subject having orsuspected of having fibrosis, wherein the subject has at least one otherdisease or condition; and administering to the subject a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a miRNA or a precursor thereof. Incertain embodiments the at least one other disease or condition ispulmonary hypertension. In certain embodiments the at least one otherdisease or condition is a blood vessel-related disease. In certainembodiments the blood-vessel related disease is selected from the groupconsisting of arterial stiffness, mediasclerosis, and arteriosclerosis.In certain embodiments the at least one other disease or condition isgut sclerosis. In certain embodiments the at least one other disease orcondition is systemic sclerosis. In certain embodiments the at least oneother disease or condition is selected from the group consisting ofretroperitoneal fibrosis, proliferative fibrosis, neoplastic fibrosis,nephrogenic systemic fibrosis, injection fibrosis, mediastinal fibrosis,myelofibrosis, post-vasectomy pain syndrome, rheumatoid arthritis.

In certain embodiments, the administering ameliorates the fibrosis. Incertain embodiments, the administering slows further progression of thefibrosis. In certain embodiments the administering halts furtherprogression of fibrosis. In certain embodiments the administeringreduces fibrosis. In certain embodiments the administering reducescollagen content.

Provided herein are methods for treating a fibroproliferative disordercomprising administering to a subject having or suspected of having afibroproliferative disorder a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary toa miRNA or a precursor thereof, thereby treating the fibroproliferativedisorder.

In certain embodiments administration comprises intravenousadministration, subcutaneous administration, pulmonary administration,intraarterial administration, or intracardiac administration.

Provided herein are methods for inhibiting fibroblast proliferationcomprising contacting a fibroblast with a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary toa miRNA or a precursor thereof, thereby inhibiting fibroblastproliferation.

Provided herein are methods for stimulating fibroblast apoptosiscomprising contacting a fibroblast with a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein thenucleobase sequence of the modified oligonucleotide is complementary tomiR-21 or a precursor thereof, thereby stimulating fibroblast apoptosis.

Provided herein are methods for increasing Sprouty 1 protein in afibroblast comprising contacting the fibroblast with a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to a miRNA or a precursor thereof,thereby stimulating Sprouty 1 protein expression.

Provided herein are compositions for inhibiting MicroRNA. In certainembodiments, the miRNA is miR-21. In certain embodiments the modifiedoligonucleotide has a nucleobase sequence complementary to a nucleobasesequence which is at least 80% identical to miR-21 or a precursorthereof. In certain embodiments miR-21 has the nucleobase sequence setforth as SEQ ID NO: 1. In certain embodiments the miR-21 precursor hasthe nucleobase sequence set forth as SEQ ID NO: 11.

In certain embodiments the modified oligonucleotide consists of 12 to 30linked nucleosides. In certain embodiments the modified oligonucleotideconsists of 12 linked nucleosides. In certain embodiments the modifiedoligonucleotide consists of 13 linked nucleosides. In certainembodiments the modified oligonucleotide consists of 14 linkednucleosides. In certain embodiments the modified oligonucleotideconsists of 15 to 24 linked nucleosides. In certain embodiments themodified oligonucleotide consists of 15 linked nucleosides. In certainembodiments the modified oligonucleotide consists of 16 linkednucleosides. In certain embodiments the modified oligonucleotideconsists of 17 linked nucleosides. In certain embodiments the modifiedoligonucleotide consists of 18 linked nucleosides. In certainembodiments the modified oligonucleotide consists of 19 linkednucleosides. In certain embodiments the modified oligonucleotideconsists of 20 linked nucleosides. In certain embodiments the modifiedoligonucleotide consists of 21 linked nucleosides. In certainembodiments the modified oligonucleotide consists of 22 linkednucleosides. In certain embodiments the modified oligonucleotideconsists of 23 linked nucleosides. In certain embodiments the modifiedoligonucleotide consists of 24 linked nucleosides.

In certain embodiments the nucleobase sequence of the modifiedoligonucleotide has no more than two mismatches to the nucleobasesequence of miR-21 or a precursor thereof. In certain embodiments thenucleobase sequence of the modified oligonucleotide has no more than onemismatch to the nucleobase sequence of miR-21 or a precursor thereof. Incertain embodiments the nucleobase sequence of the modifiedoligonucleotide has no mismatches to the nucleobase sequence of miR-21or a precursor thereof.

In certain embodiments the nucleobase sequence of the modifiedoligonucleotide comprises at least 15 contiguous nucleobases of thenucleobase sequence of SEQ ID NO: 12. In certain embodiments thenucleobase sequence of the modified oligonucleotide comprises at least16 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 12.In certain embodiments the nucleobase sequence of the modifiedoligonucleotide comprises at least 17 contiguous nucleobases of thenucleobase sequence of SEQ ID NO: 12. In certain embodiments thenucleobase sequence of the modified oligonucleotide comprises at least18 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 12.In certain embodiments the nucleobase sequence of the modifiedoligonucleotide comprises at least 19 contiguous nucleobases of thenucleobase sequence of SEQ ID NO: 12. In certain embodiments thenucleobase sequence of the modified oligonucleotide comprises at least20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 12.In certain embodiments the nucleobase sequence of the modifiedoligonucleotide comprises at least 21 contiguous nucleobases of thenucleobase sequence of SEQ ID NO: 12. In certain embodiments thenucleobase sequence of the modified oligonucleotide comprises at least22 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 12.

In certain embodiments the nucleobase sequence of the modifiedoligonucleotide consists of the nucleobase sequence of SEQ ID NO: 12.

In certain embodiments, the compound comprises a modifiedoligonucleotide conjugated to a ligand. In certain embodiments has thestructure (III)

Wherein

Each Q is independently a 2′-O-methyl modified nucleoside;x is

One of A and B is S while the other is O;y is

Each of z¹, z², z³, and z⁴ is independently x or y;n=6-17

L is

Wherein:

X is N(CO)R⁷, or NR⁷;Each of R¹, R³ and R⁹, is independently, H, OH, or —CH₂OR^(b) providedthat at least one of R¹, R³ and R⁹ is OH and at least one of R¹, R³ andR⁹ is —CH₂OR^(b);R⁷ is R^(d) or C₁-C₂₀ alkyl substituted with NR^(c)R^(d) or NHC(O)R^(d);R^(c) is H or C₁-C₆ alkyl;R^(d) is a carbohydrate radical; or a steroid radical, which isoptionally tethered to at least one carbohydrate radical; and

R^(b) is

with one of A and B is S while the other is O.

In certain embodiments, Rd is cholesterol. In certain embodiments, eachof z¹, z², z³, and z⁴ is

with one of A and B is S while the other is O.

In certain embodiments, R¹ is —CH₂OR^(b). In certain embodiments, R⁹ isOH. In certain embodiments, R¹ and R⁹ are trans. In certain embodiments,R¹ and R³ are trans. In certain embodiments, R³ is —CH₂OR^(b). Incertain embodiments, R¹ is OH. In certain embodiments, R¹ and R³ aretrans. In certain embodiments, R³ and R⁹ are trans. In certainembodiments, R⁹ is CH₂OR^(b). In certain embodiments, X is NC(O)R⁷. Incertain embodiments, R⁷ is —CH₂(CH₂)₃CH₂NHC(O)R^(d).

In certain embodiments, at least one internucleoside linkage is amodified internucleoside linkage. In certain embodiments, eachinternucleoside linkage is a modified internucleoside linkage. Incertain embodiments, at least one internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, eachinternucleoside linkage is a phosphorothioate internucleoside linkage.In certain embodiments, at least one nucleoside comprises a modifiedsugar. In certain embodiments, a plurality of nucleosides comprises amodified sugar. In certain embodiments, each nucleoside comprises amodified sugar. In certain embodiments, each nucleoside comprises a2′-O-methoxyethyl sugar. In certain embodiments, each of a plurality ofnucleosides comprises a 2′-O-methoxyethyl sugar and each of a pluralityof nucleosides comprises a 2′-fluoro sugar. In certain embodiments, eachmodified sugar is independently selected from a 2′-O-methoxyethyl sugar,a 2′-fluoro sugar, a 2′-O-methyl sugar, or a bicyclic sugar moiety. Incertain embodiments, at least one nucleoside comprises a modifiednucleobase. In certain embodiments, the modified nucleobase is a5-methylcytosine. In certain embodiments, at least one nucleosidecomprises a cytosine, wherein the cytosine is a 5-methylcytosine. Incertain embodiments, each cytosine is a 5-methylcytosine.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to the nucleobase sequenceof SEQ ID NO: 1. In certain embodiments, the nucleobase sequence of themodified oligonucleotide is at least 95% complementary to the nucleobasesequence of SEQ ID NO: 1. In certain embodiments, the nucleobasesequence of the modified oligonucleotide is 100% complementary to thenucleobase sequence of SEQ ID NO: 1. In certain embodiments, thenucleobase sequence of the modified oligonucleotide has full-lengthcomplementary to the nucleobase sequence of SEQ ID NO: 1. In certainembodiments, the nucleobase sequence of the modified oligonucleotide isat least 90% complementary to the nucleobase sequence of SEQ ID NO: 11.In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 95% complementary to the nucleobase sequenceof SEQ ID NO: 11. In certain embodiments, the nucleobase sequence of themodified oligonucleotide is 100% complementary to the nucleobasesequence of SEQ ID NO: 11. In certain embodiments, the miR-21 nucleobasesequence consists of the nucleobase sequence of SEQ ID NO: 1. In certainembodiments, the precursor nucleobase sequence consists of thenucleobase sequence of SEQ ID NO: 11.

In certain embodiments, the compound comprising the modifiedoligonucleotide is prepared as a pharmaceutical composition. In certainembodiments, the modified oligonucleotide is prepared as apharmaceutical composition.

In certain embodiments, the compound consists of a modifiedoligonucleotide. In certain embodiments, the modified oligonucleotide isa single-stranded modified oligonucleotide. In certain embodiments, themodified oligonucleotide is an antisense oligonucleotide.

Provided herein are compositions for use in the treatment, prevention,and/or amelioration of fibrosis. Further provided herein are compoundscomprising modified oligonucleotides having a nucleobase sequencecomplementary to a miRNA, for use in the treatment, prevention, and/oramelioration of fibrosis.

Provided herein are antisense oligonucleotides complementary to miR-21for the manufacture of a medicament for the treatment and/or preventionof fibrosis.

Provided herein are antisense oligonucleotides complementary to miR-21for use in the treatment and/or prevention of fibrosis.

Provided herein is the use of miR-21 and/or an antisense oligonucleotideagainst miR-21 for the treatment of fibrosis.

Provided herein is the use of miR-21 and/or an antisense oligonucleotideagainst miR-21 for the diagnosis of fibrosis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Deregulation of miR-21 in cardiac disease and predominantexpression in cardiac fibroblasts

(a) Analysis of microRNA-expression by microarrays. RNA was isolatedfrom left ventricular myocardium of a murine model of heart failure(β1AR-TG mice) at early (three months old), moderate (six months old)and late stages (twelve months) of heart failure. Expression ispresented as fold regulation vs. wild-type controls. miR-21 is marked inred. Data are from 3-4 independent hybridizations per group.

(b) Left Northern blot analysis of miR-21 expression at different stagesof heart failure in β1AR-TG mice. Right Quantitative analysis of thedata from the upper panel.

(c) Left Northern blot analysis of miR-21 expression in non-failing andfailing human left ventricular myocardium. Right Quantitative analysisof the mature form of miR-21 in non-failing and failing human leftventricular myocardium.

(d) Upper Neonatal cardiomyocytes stained with4′,6-diamidino-2-phenylindole (DAPI) and an antibody directed againstα-actinin after transfection with scrambled-miR (control-pre-miR, 50 nM,72 h), synthetic miR-21 (pre-miR-21, 50 nM, 72 h) and a miR-21 inhibitor(anti-miR-21, 50 nM, 72 h). Cardiomyocytes were cultured under controlconditions (see Supplemental Methods) or stimulated with FCS (5%) for 48h. Lower Quantitative analysis of individual cardiomyocyte sizes(histogram analysis). n>200 cardiomyocytes were analyzed per group.

(e) Upper Generation of transgenic mice expressing miR-21 under thecontrol of the alpha-MHC promoter. Middle Northern blot of mature miR-21in wild type (WT) and transgenic (TG) animals. Lower Cross sectionalcardiac areas from wild type and miR-21-transgenic mice stained todetermine general morphology (HE-stain) and collagen deposition (Siriusred).

(f) miR-21 expression in cardiac fibroblasts and cardiomyocytes.Northern blot (Upper) and quantitative real-time-PCR analysis (Lower).All error bars indicate SEM. n=3-6 for a-f.

FIG. 2 Inhibition of Sprouty1 through miR-21 derepresses ERK-signalingand enhances fibroblast survival

(a) Hearts from Spry1LacZ+/− mice were stained with X-gal. Macroscopic(upper) and microscopic (lower) analysis shows detection of LacZ incardiac fibroblasts. Short black bar represents 100 μm. Big black barrepresents 10

(b, c) Upper Detection of SPRY1, ERK1/2 and phospho-ERK1/2 in failinghuman left ventricles (b) and after transfection of co-culturedcardiomyocytes and fibroblasts with scrambled-microRNA (Pre-miR™negative Control #2, 50 nM, 72 h), synthetic miR-21 (pre-miR-21, 50 nM,72 h) or a miR-21 inhibitor (anti-miR-21, 50 nM, 72 h). LowerQuantitative analysis of Western blotting results.

(d) Upper Determination of SPRY1, ERK1/2, phospho-ERK1/2 aftersiRNA-mediated knockdown of SPRY1 (150 nM, 72 h) or treatment withscrambled siRNA (150 nM, 72 h) of the co-cultured cardiac cells. LowerQuantitative analysis of Western blotting results.

(e) Upper FACS analysis of annexin V-positive cardiac fibroblasts aftertreatment with scrambled-microRNA (control-pre-miR, 100 nM, 72 h),synthetic miR-21 (pre-miR-21, 100 nM, 72 h), miR-21 antagonists(anti-miR-21, 100 nM, 72 h) or respective controls (100 nM, 72 h). LowerQuantitative analysis of annexin V-positive cells after respectivetreatments.

(f) Upper FGF2 concentration in supernatants of cultured cardiacfibroblasts after treatment with scrambled-microRNA (control pre-miR,100 nM, 72 h), synthetic miR-21 (pre-miR-21, 100 nM, 72 h) or miR-21antagonists (anti-miR-21, 100 nM, 72 h) or (lower) after siRNA-mediatedknockdown of SPRY1 (150 nM, 72 h) or treatment with scrambled siRNA (150nM, 72 h) All error bars indicate SEM. n=3-6 for a-f.

FIG. 3 Therapeutic silencing of miR-21 in vivo prevents cardiac fibrosisand heart failure

(a) Upper Fluorescence microscopic detection of Cy3 and DAPI staining inleft ventricular heart tissue after injection of Cy3-labeled modifiedoligonucleotide via the jugular vein. Controls received PBS injections.Lower Mice subjected to transaortic constriction (TAC) or sham operationwere injected with control (PBS, 200 μl) or antagomir-21 (200 μl; 80mg/kg/d) 24 h post-operation for consecutive three days.

(b) Upper Northern blot analysis of miR-21 expression in untreated(control) and antagomir-21 treated mice after TAC. Lower Quantitativeanalysis of cardiac miR-21 expression.

(c) Upper Western blot analysis of SPRY1, ERK1/2 and phospho-ERK1/2 inmice after sham-operation or after TAC treated with either control orantagomir-21. Expression of G beta was shown as a housekeeping control.Lower Quantitative analysis of Western blot results.

(d) Cardiac sections after Sirius red staining for detection ofmyocardial fibrosis in control- and antagomir-21 treated mice.

(e) Quantitative analysis of myocardial fibrosis (left) and heart/bodyweight (right) in control- and antagomir-21 treated mice.

(f) Upper Global transcriptome analysis of the mouse genome in hearttissue of mice after sham-operation or after TAC treated with control orantagomir-21. Red (green) boxes show significantly (p<0.05) induced(repressed) genes. Lower Normalization of upregulated genes coding forproteins involved in myocardial fibrosis after TAC by antagomir-21treatment.

(g) Echocardiographic analyses. LVD, left ventricular diameter; FS,fractional shortening.

(h) Proposed mechanism underlying the derepression of cardiac ERKsignaling through miR-21-mediated inhibition of SPRY1. All error barsindicate SEM, n=3-6 for a-g.

FIG. 4 Transcriptional regulation of miR-21

(a) Sequence conservation within the miR-21-promoter regions ofdifferent species in comparison to the human miR-21-promoter. Barsindicate degree of conservation.

(b) Luciferase activity of human miR-21-promoter constructs. Progressiveshortening of the native promoter leads to identification of a 117 bpregion that is responsible for expression of miR-21. n=3.

(c) Luciferase data of human miR-21-promoter constructs afterdeletion/mutation of individual transcription factor binding sites. n=3.

FIG. 5 Knockdown of the ubiquitously expressed miR-21 induces a cardiacphenotype in zebrafish

(a) Left Lateral view of MO-control embryos and miR-21 morphants at 80hpf. A, atrium; V, ventricle; dark arrows highlight pericardial edema.

Right Upper Control-morpholino (MO-control) injected hearts displaynormal heart morphology at 80 hours post fertilization (hpf). The heartsare looped, endocardial and myocardial layers are well developed and theventricle (V) and atrium (A) is separated by the atrio-ventricular(AV)-ring. Lower miR-21 morphants (MO-1 injected) develop pericardialedema due to loss of ventricular contractility and display shortenedtails, whereas the development of other organ systems proceeds normally.

(b) Inhibition of miR-21 function by two different morpholino-modifiedantisense oligonucleotides (MO-1, n=496 and MO-2, n=460) leads toidentical phenotypes in >90% of the injected embryos. Data are from 3independent injections per group.

(c) Myocardial function displayed as fractional shortening (FS) of theventricular chamber of MO-control (n=6) and miR-21 morphants injected bytwo different morpholinos (M0-1, n=8 and MO-2, n=8) at 48, 72, 96 and120 hpf. Ventricular FS of miR-21 morphant ventricles severely decreasesover time.

FIG. 6 Transfection efficacy in cultured cardiomyocytes

(a) Cultured neonatal cardiomyocytes were transfected with Cy-3 labeledmiR-21 (50 nM, 72 h, Ambion, USA) and stained with DAPI (right). Forcomparison cultured cardiomyocytes were stained with DAPI alone (left).

(b) Northern blot analysis of miR-21 expression in culturedcardiomyocytes after transfection with scrambled-microRNA(control-pre-miR, 50 nM, 72 h), a miR-21 inhibitor (anti-miR-21, 50 nM,72 h), or synthetic miR-21 (pre-miR-21, 50 nM, 72 h).

All error bars indicate SEM; n=3-6 for a) and b).

FIG. 7 MicroRNA-binding sites within the 3′UTR of the Spry1 gene

Upper Change in expression of various microRNAs with binding siteswithin the 3′UTR of the Spry1 gene.

Lower microRNAs with binding sites within the 3′UTR analyzed by microRNAmicroarray are presented in grey. Cds=coding sequence

FIG. 8 miR-21 real-time PCR expression data

In addition to determination by Northern blotting, expression of miR-21was analyzed by real-time-PCR in left ventricular tissue of mice aftersham-operation, sham+antagomir-21 treatment, TAC and TAC+antagomir-21treatment (left), as well as in hearts from wildtype mice and miR-21transgenic mice (right). n=4-6 per group.

DATA NOT SHOWN

A screen for theoretical miR-21 targets revealed 22 known potentialtarget genes, of which 8 were shown to be expressed within cardiactissue. Combination of three different target prediction toolsidentified Spry1 (sprouty1) as a highly likely candidate.

Transcriptional Regulation of miR-21

There is sequence conservation within the miR-21-promoter regions ofdifferent species in comparison to the human miR-21-promoter. Luciferaseactivity of human miR-21-promoter constructs after deletion/mutation ofindividual transcription factor binding sites was shown. Progressiveshortening of the native promoter leads to identification of a 117 bpregion that is responsible for expression of miR-21. n=3.

Transfection Efficacy in Cultured Cardiomyocytes

Cultured neonatal cardiomyocytes were transfected with Cy-3 labeledmiR-21 (50 nM, 72 h, Ambion, USA) and stained with DAPI. For comparisoncultured cardiomyocytes were stained with DAPI alone. Northern blottingof miR-21 expression in cultured cardiomyocytes after transfection withscrambled-microRNA (control-pre-miR, 50 nM, 72 h), a miR-21 inhibitor(anti-miR-21, 50 nM, 72 h), or synthetic miR-21 (pre-miR-21, 50 nM, 72h) was analyzed.

MicroRNA-Binding Sites within the 3.UTR of the Spry1 Gene

There is a change in expression of various microRNAs with binding siteswithin the 3′UTR of the Spry1 gene. microRNAs with binding sites withinthe 3′UTR were analyzed by microRNA microarray.

miR-21 Real-Time PCR Expression Data

In addition to determination by Northern blotting, expression of miR-21was analyzed by realtime-PCR in left ventricular tissue of mice aftersham-operation, sham+miR-21 antagonist treatment, TAC and TAC+miR-21antagonist treatment as well as in hearts from wildtype mice and miR-21transgenic mice.

DETAILED DESCRIPTION Definitions

“Fibrosis” means the formation or development of excess fibrousconnective tissue in an organ or tissue. In certain embodiments,fibrosis occurs as a reparative or reactive process. In certainembodiments, fibrosis occurs in response to damage or injury. The term“fibrosis” is to be understood as the formation or development of excessfibrous connective tissue in an organ or tissue as a reparative orreactive process, as opposed to a formation of fibrous tissue as anormal constituent of an organ or tissue.

An antisense oligonucleotide is to be understood as a oligonucleotidewhich has a certain sequence complementary to another sequence, inparticular a sequence complementary to miR-21. A target of miR-21 mayalso be understood to encompass a downstream target of miR-21. It isimportant to note that an inhibition of miR-21, e.g. via anoligonucleotide with a sequence at least complementary to miR-21 willlead to a derepression or even an overexpression of targets of miR-21,like Sprouty and the like.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease or condition.

“Subject suspected of having fibrosis” means a subject exhibiting one ormore clinical indicators of fibrosis.

“Preventing” or “prevention” refers to delaying or forestalling theonset, development or progression of a condition or disease for a periodof time, including weeks, months, or years.

“Treatment” or “treat” means the application of one or more specificprocedures used for the cure or amelioration of a disease. In certainembodiments, the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“Subject in need thereof” means a subject identified as in need of atherapy or treatment.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration,” means administration through injection orinfusion.

Parenteral administration includes, but is not limited to, subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, and intracranialadministration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Intraarterial administration” means administration into an artery.

“Intracardial administration” means administration into the heart. Incertain embodiments, intracardial administration occurs by way of acatheter. In certain embodiments, intracardial administration occurs byway of open heart surgery.

“Pulmonary administration” means administration to the lungs.

“Improves liver function” means the changes liver function toward normalparameters. In certain embodiments, liver function is assessed bymeasuring molecules found in a subject's blood. For example, in certainembodiments, improved liver function is measured by a reduction in bloodliver transaminase levels.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a modifiedoligonucleotide and a sterile aqueous solution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean any nucleic acid capable of beingtargeted by antisense compounds.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid and induce adesired effect.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid to induce a desired effect. Incertain embodiments, a desired effect is reduction of a target nucleicacid.

“Modulation” means to a perturbation of function or activity. In certainembodiments, modulation means an increase in gene expression. In certainembodiments, modulation means a decrease in gene expression.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“5′ target site” refers to the nucleobase of a target nucleic acid whichis complementary to the 5′-most nucleobase of a particularoligonucleotide.

“3′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 3′-most nucleobase of a particular oligonucleotide.

“Region” means a portion of linked nucleosides within a nucleic acid. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a region of a target nucleic acid. Forexample, in certain such embodiments a modified oligonucleotide iscomplementary to a region of a miRNA stem-loop sequence. In certain suchembodiments, a modified oligonucleotide is 100% to a region of a miRNAstem-loop sequence.

“Segment” means a smaller or sub-portion of a region.

“Nucleobase sequence” means the order of contiguous nucleobases, in a 5′to 3′ orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means a first nucleobase sequence is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical, or is 100%identical, to the complement of a second nucleobase sequence over aregion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore nucleobases, or that the two sequences hybridize under stringenthybridization conditions. In certain embodiments a modifiedoligonucleotide that has a nucleobase sequence which is 100%complementary to a miRNA, or precursor thereof, may not be 100%complementary to the miRNA, or precursor thereof, over the entire lengthof the modified oligonucleotide.

“Complementarity” means the nucleobase pairing ability between a firstnucleic acid and a second nucleic acid.

“Full-length complementarity” means each nucleobase of a first nucleicacid is capable of pairing with each nucleobase at a correspondingposition in a second nucleic acid. For example, in certain embodiments,a modified oligonucleotide wherein each nucleobase has complementarityto a nucleobase in an miRNA has full-length complementarity to themiRNA.

“Percent complementary” means the number of complementary nucleobases ina nucleic acid divided by the length of the nucleic acid. In certainembodiments, percent complementarity of a modified oligonucleotide meansthe number of nucleobases that are complementary to the target nucleicacid, divided by the number of nucleobases of the modifiedoligonucleotide. In certain embodiments, percent complementarity of amodified oligonucleotide means the number of nucleobases that arecomplementary to a miRNA, divided by the number of nucleobases of themodified oligonucleotide.

“Percent region bound” means the percent of a region complementary to anoligonucleotide region. Percent region bound is calculated by dividingthe number of nucleobases of the target region that are complementary tothe oligonucleotide by the length of the target region. In certainembodiments, percent region bound is at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

“Percent identity” means the number of nucleobases in first nucleic acidthat are identical to nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

“Substantially identical” used herein may mean that a first and secondnucleobase sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical, or 100% identical, over a region of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of pairing with a nucleobase at a corresponding position of asecond nucleic acid.

“Non-complementary nucleobase” means two nucleobases that are notcapable of pairing through hydrogen bonding.

“Identical” means having the same nucleobase sequence.

“miRNA” or “miR” means a non-coding RNA between 18 and 25 nucleobases inlength which hybridizes to and regulates the expression of a coding RNA.In certain embodiments, a miRNA is the product of cleavage of apre-miRNA by the enzyme Dicer. Examples of miRNAs are found in the miRNAdatabase known as miRBase (http://microrna.sanger.ac.uk/).

“Pre-miRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which contains a miRNA. In certain embodiments, a pre-miRNAis the product of cleavage of a pri-miR by the double-strandedRNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature miRNA sequence. Pre-miRNA sequences and stem-loopsequences may overlap. Examples of stem-loop sequences are found in themiRNA database known as miRBase (http://microrna.sanger.ac.uk/).

“Pri-miRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“miRNA precursor” means a transcript that originates from a genomic DNAand that comprises a non-coding, structured RNA comprising one or moremiRNA sequences. For example, in certain embodiments a miRNA precursoris a pre-miRNA. In certain embodiments, a miRNA precursor is apri-miRNA.

“Monocistronic transcript” means a miRNA precursor containing a singlemiRNA sequence.

“Polycistronic transcript” means a miRNA precursor containing two ormore miRNA sequences.

“Seed region” means nucleotides 2 to 6 or 2 to 7 from the 5′-end of amature miRNA sequence.

“Oligomeric compound” means a compound comprising a polymer of linkedmonomeric subunits.

“Oligonucleotide” means a polymer of linked nucleosides, each of whichcan be modified or unmodified, independent from one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Natural nucleobase” means a nucleobase that is unmodified relative toits naturally occurring form.

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“miR antagonist” means a modified oligonucleotide complementary to amiRNA, or a precursor thereof. For example, “miR-X antagonist” means amodified oligonucleotide having nucleobase complementarity to miR-X. Incertain embodiments, an antagonist is a miR-21 antagonist.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar” means substitution and/or any change from a naturalsugar.

“Modified nucleobase” means any substitution and/or change from anatural nucleobase.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having aO-methoxyethyl modification at the 2′ position.

“2′-O-fluoro sugar” or “2′-F sugar” means a sugar having a fluoromodification of the 2′ position.

“Bicyclic sugar moiety” means a sugar modified by the bridging of twonon-geminal ring atoms.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification.

“2′-O-methyl” nucleoside means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety.

“Motif” means a pattern of modified and/or unmodified nucleobases,sugars, and/or internucleoside linkages in an oligonucleotide.

A “fully modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage is modified.

A “uniformly modified oligonucleotide” means each nucleobase, eachsugar, and/or each internucleoside linkage has the same modificationthroughout the modified oligonucleotide.

A “stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by 2′-deoxynucleosideslinked by phosphodiester internucleoside linkages. For example, incertain embodiments, a stabilizing modification is a stabilizingnucleoside modification. In certain embodiments, a stabilizingmodification is a internucleoside linkage modification.

A “stabilizing nucleoside” means a nucleoside modified to provideenhanced nuclease stability to an oligonucleotide, relative to thatprovided by a 2′-deoxynucleoside. In one embodiment, a stabilizingnucleoside is a 2′-modified nucleoside.

A “stabilizing internucleoside linkage” means an internucleoside linkagethat provides enhanced nuclease stability to an oligonucleotide relativeto that provided by a phosphodiester internucleoside linkage. In oneembodiment, a stabilizing internucleoside linkage is a phosphorothioateinternucleoside linkage.

Overview

It is discovered herein that modified oligonucleotides complementary tomiR-21 are pharmaceutical agents for the inhibition of miR-21. Incertain embodiments, the modified oligonucleotides are administered to asubject having a disease characterized by the upregulation of miR-21. Incertain embodiments, the disease is cancer. In certain embodiments, thedisease is heart failure. In certain embodiments, the disease isfibrosis. Fibrosis results from the development of excess fibrousconnective tissue in an organ or tissue in response to damage or injury.Left untreated, fibrosis can lead to a variety of conditions of theheart, lungs, kidney, liver, and skin, among other tissues.

It is discovered herein that fibroblast-derived miR-21 plays a criticalrole in fibrosis. Enhancement of miR-21 levels promoted fibroblastsurvival, whereas suppression of endogenous miR-21 induced apoptoticcell death. Thus, herein identified is a mechanism by which miR-21regulates fibroblast survival. Aberrant fibroblast proliferation andsurvival may lead to fibrosis. Accordingly, modified oligonucleotidescomplementary to miR-21 are pharmaceutical agents for the treatment offibrosis. In certain embodiments, a modified oligonucleotidecomplementary to miR-21 is an antisense oligonucleotide complementary tomiR-21.

The inventors demonstrate a critical role of fibroblast-derived miR-21and SPRY1 in the heart (FIG. 3h ). The data suggest that stress-inducedand miRNA-mediated activation of ERK-MAPkinase activity may importantlyregulate cardiac fibrosis. This study represents the first example of amicroRNA therapeutic application in a disease model. Specificallyantagonizing miR-21 prevented the structural and functionaldeterioration in a murine model. These findings suggest a newtherapeutic entry point for heart failure and show the broad therapeuticpotential of microRNA antagonists.

In one aspect the present invention relates to the use of miR-21, anantisense oligonucleotide against miR-21 and/or a target of miR-21 forthe manufacture of a medicament for the treatment and/or prevention offibrosis and/or fibrosis related diseases.

The invention particularly concerns cardiac specific diseases involvingfibrosis (=fibrosis related diseases), like cardiac hypertrophy,hypertensive heart disease, diastolic and systolic heart failure,storage-diseases related to heart, such as M. Fabry, cardiomyopathies,e.g. dilatative cardiomyopathy, hypertrophic cardio-myopathy with andwithout obstruction, restrictive cardiomyopathy, arrhythmogenic rightventricular cardiomyopathy and other forms of cardiomyopathy, like e.g.diabetic cardiomyopathy, constrictive pericarditis, coronary arterydisease, myocardial infarction, acute and chronic right heart failure,cardiac arrhythmias due to fibrosis, myocarditis-related fibrosis,diseases of the heart valves leading to valve stenosis or insufficiency(e.g. sclerosis), e.g. mitral valve stenosis and/or insufficiency,aortic valve stenosis and/or insufficiency, tricuspidal valve stenosisand/or insufficiency, pulmonary valve stenosis and/or insufficiency.

In a further aspect the invention concerns other diseases involvingfibrosis (=fibrosis related diseases), not related to the cardiacsystem. Non-limiting examples are lung fibrosis, chronic obstructivelung diseases, pulmonary hypertension, liver fibrosis due to a toxicsurrounding, hepatitis and/or secondary to right heart failure, skinfibrosis, e.g. development of keloids after injury, blood vessel-relateddiseases, such as arterial stiffness related to age or hypertension,mediasclerosis, arteriosclerosis, age-related fibrosis of differentorgans, gut sclerosis, e.g. during Crohn's disease, systemic sclerosisand CREST syndrome etc., kidney fibrosis, neoplastic fibrosis and/orrheumatoid arthritis.

Further well-known fibrosis-related diseases and disorders are e.g.endomyocardial fibrosis and idiopathic myocardiopathy, cirrhosis whichcan result from fibrosis of the liver, idiopathic pulmonary fibrosis ofthe lung, diffuse paren-chymal lung disease, mediastinal fibrosis,myelofibrosis, post-vasectomy pain syndrome, retroperitoneal fibrosisand nephrogenic systemic fibrosis.

All aspects of the invention, in particular the aspects mentioned in theaccompanying claims may be used for the diagnosis, treatment and/orprevention of the above mentioned diseases, disorders and conditions,which are given as possible examples. The list is not limiting.

In one embodiment, the invention relates to strategies for modulatingmiR-21. In another embodiment, the invention relates to strategies formodulating, e.g. overexpressing and/or upregulating targets of miR-21.Possible modulations are implantable devices, like viral vectors andliposomal formulations, etc, gene transfer systems, sponges and thelike.

Sprouty (SPRY1) is herein identified as a target of miR-21. Both miR-21and SPRY1 are expressed in cardiac fibroblasts, among other cell types.Increasing miR-21 expression in cardiac fibroblasts induced a strongrepression of SPRY protein expression and further increasedERK-MAPkinase activation. In a preferred embodiment the target isselected from the group consisting of Sprouty1 (SPRY1), in particularSEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 andRtn4.

In one aspect the present invention relates to an antisenseoligonucleotide against (or complementary to) SEQ ID NO: 2 to SEQ ID NO:4, Sprouty1 (SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi,Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 for the diagnosis, treatmentand/or prevention of fibrosis and/or fibrosis related diseases. Incertain aspects, an antisense oligonucleotide complementary to a targetselected from the group consisting of Sprouty1 (SPRY1), in particularSEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1and/or Rtn4, interferes with the ability of a microRNA, such as miR-21,to bind to a target site and inhibit expression of the selected target.In certain aspects, such an antisense oligonucleotide comprises one ormore nucleoside modifications.

In one aspect the present invention relates to a cell deficient formiR-21, SEQ ID NO: 2 to SEQ ID NO: 4, Sprouty1 (SPRY1), in particularSEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1and/or Rtn4.

In one aspect the invention relates to a non-human mammal knock-outorganism deficient for miR-21, SEQ ID NO: 2 to SEQ ID NO: 4, Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1,Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 as a disease model for fibrosisand/or fibrosis related diseases. Also encompassed are transgenicanimals, in particular non-human mammals which overexpress miR-21.

It is discovered herein that fibroblast-derived miR-21 contributes toheart failure. Analysis of left ventricular cardiac tissue samples fromsubjects with end-stage heart failure due to idiopathic dilatedcardiomyopathy revealed increased miR-21 expression and repressed SPRY1protein expression. Additionally, ERK-MAPkinase was activated in samplesfrom these subjects, as evidenced by an increased phosphor-ERK/ERKratio. It is discovered herein that in an animal model of heart failurethe administration of a modified oligonucleotide complementary to miR-21resulted in significant attenuation of cardiac fibrosis. Heart failureis characterized by, among other parameters, cardiac fibrosis.Accordingly, modified oligonucleotides complementary to miR-21 arepharmaceutical agents for the treatment of cardiac fibrosis.

It is also discovered herein that in an animal model of heart failure,the attenuation of cardiac fibrosis is accompanied by attenuation ofheart weight increases. Further, assessment of cardiac function byechocardiography revealed that the administration of a modifiedoligonucleotide complementary to miR-21 prevented left ventriculardilation and normalized parameters of fractional shortening. Heartfailure may be characterized by, among other parameters, heart weightincreases, left ventricular dilation, and impairment of fractionalshortening. Accordingly, modified oligonucleotides complementary tomiR-21 are therapeutic agents for the treatment, amelioration, andprevention of heart failure associated with cardiac fibrosis.

In one aspect the present invention relates to a promoter region of amicroRNA (miRNA) comprising a modification of a calcium/cAMP responseelement protein (CREB) and/or serum response factor (SRF) binding sitefor diagnosis, prevention and/or therapy of fibrosis and/or fibrosisrelated diseases.

In one embodiment, the promoter region, the gene of mirR-21 itselfand/or the 3′UTR of various messenger RNAs may contain polymorphisms,mutations, in particular point mutations, deletions, truncations and/orinversion. All these amendments to the wild-type sequence lead to the denovo formation of a novel miR-21 binding site or to the deletion of amiR-21 binding site. In another embodiment the modification of thepromoter region is selected from the group consisting of a pointmutation, a truncation, a deletion and an inversion. Further, thepromoter region can be selected from the group consisting of SEQ ID NO:2 to SEQ ID NO: 4.

Diagnostic Applications

In one aspect the present invention relates to the use of miR-21, anantisense oligonucleotide against miR-21 and/or a target of miR-21 forthe diagnosis of fibrosis and/or fibrosis related diseases. An antisenseoligonucleotide is to be understood as a oligonucleotide which has acertain sequence complementary to another sequence, in particular asequence complementary to miR-21. A target of miR-21 may also beunderstood to encompass a downstream target of miR-21. It is importantto note that an inhibition of miR-21, e.g. via an oligonucleotide with asequence at least complementary to miR-21 will lead to a derepression oreven an overexpression of targets of miR-21, like Sprouty and the like.

In one aspect the invention relates a method for diagnosing fibrosisand/or fibrosis related diseases comprising the steps of:

(a) providing a sample of an patient supposed to suffer from fibrosisand/or fibrosis related diseases;(b) measuring the expression of miR-21, Sprouty1 (SPRY1), in particularSEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1and/or Rtn4;

wherein an elevated level of miR-21 and/or a reduced level of Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1,Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 in comparison to a control sampleindicates fibrosis and/or fibrosis related diseases or a predispositionthereof.

In one aspect the present invention relates to a method for screening ofa pharmaceutically active compound for the treatment and/or theprevention of fibrosis and/or fibrosis related diseases or apredisposition thereof, comprising the steps of:

(a) Providing a sample containing miR-21, Sprouty1 (SPRY1), inparticular SEQ ID NO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl,Nfib, Lnx1 and/or Rtn4;(b) Contacting a candidate substance with the sample;(c) Determining the effect of the candidate substance on the sample;wherein an alteration of miR-21, Sprouty1 (SPRY1), in particular SEQ IDNO: 7 or SEQ ID NO: 8, Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4indicates a pharmaceutically active compound.

Certain Diseases and Conditions

Provided herein are methods for treating a subject having or suspectedof having fibrosis. Also provided are methods for treating a subjectidentified as having or suspected of having fibrosis. In certainembodiments, such methods comprise administering to a subject a modifiedoligonucleotide having a nucleobase sequence complementary to a miRNA ora precursor thereof. In certain embodiments, the miRNA is miR-21.

Further provided herein are methods for preventing fibrosis in a subjectat risk for developing fibrosis. In certain embodiments, such methodscomprise administering to a subject at risk for developing fibrosis amodified oligonucleotide having a nucleobase sequence complementary to amiRNA or a precursor thereof. In certain embodiments, the miRNA ismiR-21.

In certain embodiments, the fibrosis is liver fibrosis. In certainembodiments, the fibrosis is lung fibrosis. In certain embodiments thefibrosis is skin fibrosis. In certain embodiments the fibrosis iscardiac fibrosis. In certain embodiments the fibrosis is kidneyfibrosis. In certain embodiments the fibrosis is lung fibrosis. Incertain embodiments the fibrosis is age-related fibrosis. In certainembodiments the fibrosis is spleen fibrosis.

In certain embodiments, the subject having or suspected of havingfibrosis has at least one cardiac disease or condition. In certainembodiments, a subject identified as having or suspected of havingfibrosis has at least one cardiac disease or condition. In certainembodiments, such methods comprise administering to a subject a modifiedoligonucleotide having a nucleobase sequence complementary to a miRNA ora precursor thereof. In certain embodiments, the miRNA is miR-21.

In certain embodiments, a cardiac disease or condition is cardiachypertrophy. In certain embodiments, a cardiac disease or condition iscardiomyopathy. In certain embodiments, cardiomyopathy is dilatativecardiomyopathy, hypertrophic cardiomyopathy with obstruction,hypertrophic cardiomyopathy without obstruction, restrictivecardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, ordiabetic cardiomyopathy.

In certain embodiments, a cardiac disease or condition is coronaryartery disease. In certain embodiments, a cardiac disease or conditionis heart-related storage disease, constrictive pericarditis, acutemyocardial infarction, chronic myocardial infarction, cardiacarrhythmia, or myocarditis-related fibrosis.

In certain embodiments, a cardiac disease or condition is heart failure.In certain embodiments, heart failure is hypertensive heart failure,diastolic heart failure, systolic heart failure, or right heart failure.

In certain embodiments, a cardiac disease or condition is heart valvedisease. In certain embodiments, heart valve disease is mitral valvestenosis, aortic valve stenosis, tricuspidal valve stenosis, orpulmonary valve stenosis. In certain embodiments, the heart valvedisease is mitral valve insufficiency, aortic valve insufficiency,tricuspidal valve insufficiency, or pulmonary valve insufficiency.

Provided herein are methods for treating a subject having or suspectedof having fibrosis, wherein the subject has a liver disease orcondition. In certain embodiments, a subject identified as having orsuspected of having fibrosis has at least one liver disease orcondition. In certain embodiments, such methods comprise administeringto a subject a modified oligonucleotide having a nucleobase sequencecomplementary to a miRNA or a precursor thereof. In certain embodiments,the miRNA is miR-21. In certain embodiments, a liver disease orcondition is chronic liver injury. In certain embodiments, a liverdisease or condition is hepatitis virus infection. In certainembodiments, a hepatitis infection is hepatitis C virus infection. Incertain embodiments a liver disease or condition is non-alcoholicsteatohepatitis. In certain embodiments a liver disease or condition iscirrhosis.

Provided herein are methods for the treatment of a subject having orsuspected of having fibrosis, wherein the subject has at least one lungdisease or condition. Also provided herein are methods for the treatmentof a subject identified as having or suspected of having fibrosis,wherein the subject has at least one lung disease or condition. Incertain embodiments, such methods comprise administering to a subject amodified oligonucleotide having a nucleobase sequence which iscomplementary to a miRNA or a precursor thereof. In certain embodiments,the miRNA is miR-21. In certain embodiments a lung disease or conditionis chronic obstructive lung disease.

Provided herein are methods for the treatment of a subject having orsuspected of having fibrosis, wherein the subject has at least one otherdisease or condition. Also provided herein are methods for the treatmentof a subject identified as having or suspected of having fibrosis,wherein the subject has at least one other disease or condition. Incertain embodiments, such methods comprise administering to a subject amodified oligonucleotide having a nucleobase sequence which iscomplementary to a miRNA or a precursor thereof. In certain embodiments,the miRNA is miR-21. In certain embodiments one other disease orcondition is pulmonary hypertension. In certain embodiments one otherdisease or condition is a blood vessel-related disease. In certain suchembodiments a blood-vessel related disease is arterial stiffness,mediasclerosis, or arteriosclerosis. In certain embodiments one otherdisease or condition is gut sclerosis. In certain embodiments anotherdisease or condition is systemic sclerosis. In certain embodiments oneother disease or condition is retroperitoneal fibrosis, proliferativefibrosis, neoplastic fibrosis, nephrogenic systemic fibrosis, injectionfibrosis, mediastinal fibrosis, myelofibrosis, post-vasectomy painsyndrome, or rheumatoid arthritis.

Provided herein are methods for treating a subject having afibroproliferative disorder. In certain embodiments such methodscomprise administering to a subject having or suspected of having afibroproliferative disorder a modified oligonucleotide having anucleobase sequence which is complementary to a miRNA or a precursorthereof. In certain embodiments, the miRNA is miR-21.

The invention additionally relates to miRNA-21 based treatments forcancer that involve targeting specific cancers by modulation ofSPRY-1-mediated ERK signalling in cancer cells. It is known that miR-21is overexpressed in many different cancer types including esophageal,colon adenocarcinoma, breast cancer, gliomas, glioblastomas, ovariancancer, hepatocellular cancer, head and neck cancer, chronic lymphocyticleukemia, pancreatic cancer, just to name some of them.

Therefore, the invention also relates to the diagnosis, preventionand/or therapy of the above mentioned cancer types. All featuresmentioned in the claims may be combined with the disclosure of thesecancer types.

Certain Routes of Administration

In certain embodiments, administering to a subject comprises parenteraladministration. In certain embodiments, administering to a subjectcomprises intravenous administration. In certain embodiments,administering to a subject comprises subcutaneous administration.

In certain embodiments, administering to a subject comprisesintraarterial administration. In certain embodiments, administering to asubject comprises intracardial administration. Suitable means forintracardial administration include the use of a catheter, oradministration during open heart surgery.

In certain embodiments, administration includes pulmonaryadministration. In certain embodiments, pulmonary administrationcomprises delivery of aerosolized oligonucleotide to the lung of asubject by inhalation. Following inhalation by a subject of aerosolizedoligonucleotide, oligonucleotide distributes to cells of both normal andinflamed lung tissue, including alveolar macrophages, eosinophils,epithelium, blood vessel endothelium, and bronchiolar epithelium. Asuitable device for the delivery of a pharmaceutical compositioncomprising a modified oligonucleotide includes, but is not limited to, astandard nebulizer device. Formulations and methods for modulating thesize of droplets using nebulizer devices to target specific portions ofthe respiratory tract and lungs are well known to those skilled in theart. Additional suitable devices include dry powder inhalers or metereddose inhalers.

In certain embodiments, pharmaceutical compositions are administered toachieve local rather than systemic exposures. For example, pulmonaryadministration delivers a pharmaceutical composition to the lung, withminimal systemic exposure.

Additional suitable administration routes include, but are not limitedto, oral, rectal, transmucosal, intestinal, enteral, topical,suppository, intrathecal, intraventricular, intraperitoneal, intranasal,intraocular, intramuscular, intramedullary, and intratumoral.

Certain Clinical Outcomes

In certain embodiments, the methods herein provide a clinicallydesirable outcome to a subject having or suspected of having fibrosis.

In certain embodiments, a clinically desirable outcome is theamelioration of heart weight increase. In certain such embodiments, aclinically desirable outcome is the amelioration of left ventriculardilation. In certain embodiments a clinically desirable outcome is theamelioration of impaired fractional shortening. In certain embodiments aclinically desirable outcome is the prevention of heart weight increase.In certain such embodiments, a clinically desirable outcome is theprevention of left ventricular dilation. In certain embodiments aclinically desirable outcome is the prevention of impaired fractionalshortening.

In certain embodiments a clinically desirable outcome is improvedcardiac function.

In certain embodiments a clinically desirable outcome is theamelioration of fibrosis. In certain embodiments a clinically desirableoutcome is the slowing of further progression of fibrosis. In certainembodiments a clinically desirable outcome is the halting of furtherprogression of fibrosis. In certain embodiments a clinically desirableoutcome is a reduction in fibrosis. In certain embodiments a clinicallydesirable outcome is a reduction in collagen content.

In certain embodiments a therapeutically desirable outcome is improvedliver function. Liver function may be assessed by liver function tests,which measure, among other things, blood levels of liver transaminases.In certain embodiments, a subject having abnormal liver function haselevated blood liver transaminases. Blood liver transaminases includealanine aminotransferase (ALT) and aspartate aminotransferase (AST). Incertain embodiments, a subject having abnormal liver function haselevated blood bilirubin. In certain embodiments, a subject has abnormalblood albumin levels. In certain embodiments, the methods providedherein alter ALT, AST, bilirubin and/or albumin levels in the blood suchthat one or more of these levels is closer to normal limits.

In certain embodiments, a subject's liver function is assessed by theChild-Pugh classification system, which defines three classes of liverfunction. In this classification system, points are assigned tomeasurements in one of five categories: bilirubin levels, albuminlevels, prothrombin time, ascites, and encephalopathy. One point isassigned per each of the following characteristics present: bloodbilirubin of less than 2.0 mg/dl; blood albumin of greater than 3.5mg/dl; a prothrombin time of less than 1.7 international normalizedratio (INR); ascites is absent; or encephalopathy is absent. Two pointsare assigned per each of the following characteristics present: bloodbilirubin of 2-3 mg/dl; blood bilirubin of 3.5 to 2.8 mg/dl; prothrombintime of 1.7-2.3 INR; ascites is mild to moderate; or encephalopathy ismild. Three points are assigned per each of the followingcharacteristics present: bilirubin of greater than 3.0 mg/dl; bloodalbumin of less than 2.8 mg/dl; prothrombin time of greater than 2.3INR; ascites is severe to refractory; or encephalopathy is severe. Thescores are added and Class A is assigned for a score of 5-6 points,Class B is assigned for a score of 7-9 points, and Class C is assignedfor a score of 10-15 points. In certain embodiments, the methodsprovided herein result in an improvement in liver function as measuredby the Child-Pugh classification system.

Certain Cellular Phenotypes

Provided herein are methods for inhibiting fibroblast cellproliferation. Also provided herein are methods for stimulatingapoptosis in a fibroblast cell. Further provided herein are methods forincreasing Sprouty 1 protein in a fibroblast cell. In certainembodiments, such methods comprise contacting a fibroblast with acompound comprising a modified oligonucleotide and having a nucleobasesequence which is complementary to a miRNA or a precursor thereof. Incertain embodiments, the miRNA is miR-21.

In certain embodiments, the fibroblast cell is in vitro. In certainembodiments, the fibroblast cell is in vivo. In certain embodiments, thecontacting occurs in vitro. In certain embodiments, the contactingoccurs in vivo. In certain embodiments, the contacting occurs ex vivo.

Certain Additional Therapies

Treatments for fibrosis may comprise more than one therapy. As such, incertain embodiments provided herein are methods for treating a subjecthaving or suspected of having fibrosis comprising administering at leastone therapy in addition to administering a modified oligonucleotidehaving a nucleobase sequence complementary to a miRNA or a precursorthereof.

In certain embodiments, the methods provided herein compriseadministering one or more additional pharmaceutical agents. In certainembodiments, additional pharmaceutical agents include, but are notlimited to, diuretics (e.g. sprionolactone, eplerenone, furosemide),inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators,angiotensin II converting enzyme (ACE) inhibitors (e.g. are captopril,enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril),angiotensin II receptor blockers (ARB) (e.g. candesartan, irbesartan,olmesartan, losartan, valsartan, telmisartan, eprosartan), calciumchannel blockers, isosorbide dinitrate, hydralazine, nitrates (e.g.isosorbide mononitrate, isosorbide dinitrate), hydralazine,beta-blockers (e.g. carvedilol, metoprolol), and natriuretic peptides(e.g. nesiritide).

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain such embodiments, the additional therapy is selected to treator ameliorate a side effect of one or more pharmaceutical compositionsof the present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, renalfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

In certain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at different times. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areprepared together in a single formulation. In certain embodiments, oneor more pharmaceutical compositions of the present invention and one ormore other pharmaceutical agents are prepared separately.

Certain Pharmaceutical Compositions

In certain embodiments, a compound comprising a modified oligonucleotidecomplementary to a miRNA, or precursor thereof, described herein isprepared as a pharmaceutical composition for the treatment of fibrosis.In certain embodiments, a compound comprising a modified oligonucleotidehaving a nucleobase sequence complementary to a miRNA or a precursorthereof is prepared as a pharmaceutical composition for the preventionof fibrosis.

In certain embodiments, a pharmaceutical composition of the presentinvention is administered in the form of a dosage unit (e.g., tablet,capsule, bolus, etc.). In certain embodiments, such pharmaceuticalcompositions comprise a modified oligonucleotide in a dose selected from25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg,125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg,170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg,215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg,260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg,305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg,350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg,395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg,440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg,485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg,530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg,575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg,620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg,665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg,710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg,755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg,and 800 mg. In certain such embodiments, a pharmaceutical composition ofthe present invention comprises a dose of modified oligonucleotideselected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of a modifiedoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. The lyophilized drug productmay be packaged in a 2 mL Type I, clear glass vial (ammoniumsulfate-treated), stoppered with a bromobutyl rubber closure and sealedwith aluminum FLIP-OFF® overseal.

In certain embodiments, the compositions of the present invention mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions; alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared using known techniques, including, but not limitedto mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

In certain embodiments, a pharmaceutical composition of the presentinvention is a liquid (e.g., a suspension, elixir and/or solution). Incertain of such embodiments, a liquid pharmaceutical composition isprepared using ingredients known in the art, including, but not limitedto, water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents.

In certain embodiments, a pharmaceutical composition of the presentinvention is a solid (e.g., a powder, tablet, and/or capsule). Incertain of such embodiments, a solid pharmaceutical compositioncomprising one or more oligonucleotides is prepared using ingredientsknown in the art, including, but not limited to, starches, sugars,diluents, granulating agents, lubricants, binders, and disintegratingagents.

In certain embodiments, a pharmaceutical composition of the presentinvention is formulated as a depot preparation. Certain such depotpreparations are typically longer acting than non-depot preparations. Incertain embodiments, such preparations are administered by implantation(for example subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a delivery system. Examples of delivery systemsinclude, but are not limited to, liposomes and emulsions. Certaindelivery systems are useful for preparing certain pharmaceuticalcompositions including those comprising hydrophobic compounds. Incertain embodiments, certain organic solvents such as dimethylsulfoxideare used.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises one or more tissue-specific delivery moleculesdesigned to deliver the one or more pharmaceutical agents of the presentinvention to specific tissues or cell types. For example, in certainembodiments, pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a co-solvent system. Certain of such co-solventsystems comprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a sustained-release system. A non-limiting exampleof such a sustained-release system is a semi-permeable matrix of solidhydrophobic polymers. In certain embodiments, sustained-release systemsmay, depending on their chemical nature, release pharmaceutical agentsover a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared for oral administration. In certain of suchembodiments, a pharmaceutical composition is formulated by combining oneor more compounds comprising a modified oligonucleotide with one or morepharmaceutically acceptable carriers. Certain of such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In certain embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certainembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In certain embodiments, pharmaceutical compositionsare formed to obtain tablets or dragee cores. In certain embodiments,disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Certain of such pharmaceuticalcompositions for inhalation are prepared in the form of an aerosol sprayin a pressurized pack or a nebulizer. Certain of such pharmaceuticalcompositions comprise a propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In certain embodiments using a pressurized aerosol,the dosage unit may be determined with a valve that delivers a meteredamount. In certain embodiments, capsules and cartridges for use in aninhaler or insufflator may be formulated. Certain of such formulationscomprise a powder mixture of a pharmaceutical agent of the invention anda suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared forrectal administration, such as a suppositories or retention enema.Certain of such pharmaceutical compositions comprise known ingredients,such as cocoa butter and/or other glycerides.

In certain embodiments, a pharmaceutical composition is prepared fortopical administration. Certain of such pharmaceutical compositionscomprise bland moisturizing bases, such as ointments or creams.Exemplary suitable ointment bases include, but are not limited to,petrolatum, petrolatum plus volatile silicones, and lanolin and water inoil emulsions. Exemplary suitable cream bases include, but are notlimited to, cold cream and hydrophilic ointment.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a modified oligonucleotide in a therapeuticallyeffective amount. In certain embodiments, the therapeutically effectiveamount is sufficient to prevent, alleviate or ameliorate symptoms of adisease or to prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides of thepresent invention is formulated as a prodrug. In certain embodiments,upon in vivo administration, a prodrug is chemically converted to thebiologically, pharmaceutically or therapeutically more active form of amodified oligonucleotide. In certain embodiments, prodrugs are usefulbecause they are easier to administer than the corresponding activeform. For example, in certain instances, a prodrug may be morebioavailable (e.g., through oral administration) than is thecorresponding active form. In certain instances, a prodrug may haveimproved solubility compared to the corresponding active form. Incertain embodiments, prodrugs are less water soluble than thecorresponding active form. In certain instances, such prodrugs possesssuperior transmittal across cell membranes, where water solubility isdetrimental to mobility. In certain embodiments, a prodrug is an ester.In certain such embodiments, the ester is metabolically hydrolyzed tocarboxylic acid upon administration. In certain instances the carboxylicacid containing compound is the corresponding active form. In certainembodiments, a prodrug comprises a short peptide (polyaminoacid) boundto an acid group. In certain of such embodiments, the peptide is cleavedupon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Compounds

In certain embodiments, the methods provided herein compriseadministration of a compound comprising a modified oligonucleotide. Incertain embodiments, the compound consists of a modifiedoligonucleotide.

In certain such embodiments, a compound comprises a modifiedoligonucleotide hybridized to a complementary strand, i.e. a compoundcomprises a double-stranded oligomeric compound. In certain embodiments,the hybridization of a modified oligonucleotide to a complementarystrand forms at least one blunt end. In certain such embodiments, thehybridization of a modified oligonucleotide to a complementary strandforms a blunt end at each terminus of the double-stranded oligomericcompound. In certain embodiments, a terminus of a modifiedoligonucleotide comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of the complementarystrand. In certain embodiments, the one or more additional nucleosidesare at the 5′ terminus of a modified oligonucleotide. In certainembodiments, the one or more additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain embodiments, at leastone nucleobase of a nucleoside of the one or more additional nucleosidesis complementary to the target RNA. In certain embodiments, eachnucleobase of each one or more additional nucleosides is complementaryto the target RNA. In certain embodiments, a terminus of thecomplementary strand comprises one or more additional linked nucleosidesrelative to the number of linked nucleosides of a modifiedoligonucleotide. In certain embodiments, the one or more additionallinked nucleosides are at the 3′ terminus of the complementary strand.In certain embodiments, the one or more additional linked nucleosidesare at the 5′ terminus of the complementary strand. In certainembodiments, two additional linked nucleosides are linked to a terminus.In certain embodiments, one additional nucleoside is linked to aterminus.

In certain embodiments, a compound comprises a modified oligonucleotideconjugated to one or more moieties which enhance the activity, cellulardistribution or cellular uptake of the resulting antisenseoligonucleotides. In certain such embodiments, the moiety is acholesterol moiety or a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In certain embodiments, a conjugategroup is attached directly to a modified oligonucleotide. In certainembodiments, a conjugate group is attached to a modified oligonucleotideby a linking moiety selected from amino, hydroxyl, carboxylic acid,thiol, unsaturations (e.g., double or triple bonds),8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA),substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl,and substituted or unsubstituted C2-C10 alkynyl. In certain suchembodiments, a substituent group is selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Certain Nucleobase Sequences

Provided herein are methods for the treatment or prevention of fibrosis.In certain embodiments, the methods comprise administration of apharmaceutical composition comprising a modified oligonucleotide. Incertain embodiments, the methods comprise administration of a compoundcomprising a modified oligonucleotide. In certain embodiments, amodified oligonucleotide has a sequence that is complementary to a miRNAor a precursor thereof. In certain embodiments, the miRNA is miR-21.

Nucleobase sequences of mature miRNAs and their corresponding stem-loopsequences described herein are the sequences found in miRBase, an onlinesearchable database of miRNA sequences and annotation, found athttp://microrna.sanger.ac.uk/. Entries in the miRBase Sequence databaserepresent a predicted hairpin portion of a miRNA transcript (thestem-loop), with information on the location and sequence of the maturemiRNA sequence. The miRNA stem-loop sequences in the database are notstrictly precursor miRNAs (pre-miRNAs), and may in some instancesinclude the pre-miRNA and some flanking sequence from the presumedprimary transcript. The miRNA nucleobase sequences described hereinencompass any version of the miRNA, including the sequences described inRelease 10.0 of the miRBase sequence database and sequences described inany earlier Release of the miRBase sequence database. A sequencedatabase release may result in the re-naming of certain miRNAs. Asequence database release may result in a variation of a mature miRNAsequence. The compounds of the present invention encompass modifiedoligonucleotides that are complementary any nucleobase sequence versionof the miRNAs described herein.

It is understood that any nucleobase sequence set forth herein isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. It is further understood that a nucleobasesequence comprising U's also encompasses the same nucleobase sequencewherein ‘U’ is replaced by ‘T’ at one or more positions having ‘U.”Conversely, it is understood that a nucleobase sequence comprising T'salso, encompasses the same nucleobase sequence wherein ‘T; is replacedby ‘U’ at one or more positions having ‘T.”

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a miRNA or a precursor thereof,meaning that the nucleobase sequence of a modified oligonucleotide is aleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identicalto the complement of a miRNA or precursor thereof over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases,or that the two sequences hybridize under stringent hybridizationconditions. Accordingly, in certain embodiments the nucleobase sequenceof a modified oligonucleotide may have one or more mismatched basepairswith respect to its target miRNA or target miRNA precursor sequence, andis capable of hybridizing to its target sequence. In certainembodiments, a modified oligonucleotide has a nucleobase sequence thatis 100% complementary to a miRNA or a precursor thereof. In certainembodiments, the nucleobase sequence of a modified oligonucleotide hasfull-length complementary to a miRNA.

In certain embodiments, a miR-21 has the nucleobase sequence5′-UAGCUUAUCAGACUGAUGUUGA-3′ (SEQ ID NO: 1). In certain embodiments amiR-21 stem-loop sequence has the nucleobase sequence5′-UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA-3′ (SEQ ID NO: 11).

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to the nucleobase sequence of miR-21 set forth as SEQID NO: 1.

In certain embodiments, a modified oligonucleotide has a sequence thatis complementary to the nucleobase sequence of a miRNA stem-loopsequence set forth as SEQ ID NO: 11. In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to theregion of nucleobases 8-29 of SEQ ID NO: 11. In certain embodiments amodified oligonucleotide has a sequence that is complementary to theregion of nucleobases 46 to 66 of SEQ ID NO: 11.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence comprising the nucleobase sequence 5′-UCAACAUCAGUCUGAUAAGCUA-3′(SEQ ID NO: 12).

In certain embodiments, a modified oligonucleotide has a nucleobasesequence consisting of the nucleobase sequence set forth as SEQ ID NO:12.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence of a pri-miRsequence comprising miR-21.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to the nucleobase sequence set forth in SEQ ID NO: 1. Incertain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least98% identity to the nucleobase sequence set forth in SEQ ID NO: 1.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to a nucleobase sequence having at least80% identity to a nucleobase sequence of a miR-21 stem-loop sequence setforth in SEQ ID NO: 11. In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence that is complementary to anucleobase sequence having at least 85%, at least 90%, at least 92%, atleast 94%, at least 96%, or at least 98% identity to a nucleobasesequence of a miR-21 stem-loop sequence set forth in SEQ ID NO: 11.

In certain embodiments, a nucleobase sequence of a modifiedoligonucleotide has full-length complementary to a miRNA nucleobasesequence listed herein, or a precursor thereof. In certain embodiments,a modified oligonucleotide has a nucleobase sequence having one mismatchwith respect to the nucleobase sequence of the mature miRNA, or aprecursor thereof. In certain embodiments, a modified oligonucleotidehas a nucleobase sequence having two mismatches with respect to thenucleobase sequence of the miRNA, or a precursor thereof. In certain,such embodiments, a modified oligonucleotide has a nucleobase sequencehaving no more than two mismatches with respect to the nucleobasesequence of the mature miRNA, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of the mature miRNA towhich it is complementary.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of the mature miRNA to which itis complementary. In certain such embodiments, the number of linkednucleosides of a modified oligonucleotide is one less than the length ofthe mature miRNA to which it is complementary. In certain suchembodiments, a modified oligonucleotide has one less nucleoside at the5′ terminus. In certain such embodiments, a modified oligonucleotide hasone less nucleoside at the 3′ terminus. In certain such embodiments, amodified oligonucleotide has two fewer nucleosides at the 5′ terminus.In certain such embodiments, a modified oligonucleotide has two fewernucleosides at the 3′ terminus. A modified oligonucleotide having anumber of linked nucleosides that is less than the length of the miRNA,wherein each nucleobase of a modified oligonucleotide is complementaryto each nucleobase at a corresponding position in a miRNA, is consideredto be a modified oligonucleotide having a nucleobase sequence 100%complementary to a portion of a miRNA sequence.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of the miRNA to which it iscomplementary. In certain such embodiments, the nucleobase of anadditional nucleoside is complementary to a nucleobase of a miRNAstem-loop sequence. In certain embodiments, the number of linkednucleosides of a modified oligonucleotide is one greater than the lengthof the miRNA to which it is complementary. In certain such embodiments,the additional nucleoside is at the 5′ terminus of a modifiedoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of a modified oligonucleotide. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is two greater than the length of the miRNA to which itis complementary. In certain such embodiments, the two additionalnucleosides are at the 5′ terminus of a modified oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of a modified oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of a modified oligonucleotide.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is 100% complementary to the nucleobasesequence of the miRNA, but the modified oligonucleotide is not 100%complementary over its entire length. In certain such embodiments, thenumber of nucleosides of a modified oligonucleotide having a 100%complementary portion is greater than the length of the miRNA. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 23 are each complementaryto a corresponding position of a miRNA that is 23 nucleobases in length,has a 23 nucleoside portion that is 100% complementary to the nucleobasesequence of the miRNA and approximately 96% overall complementarity tothe nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of a modifiedoligonucleotide is 100% complementary to a portion of the nucleobasesequence of a miRNA. For example, a modified oligonucleotide consistingof 22 linked nucleosides, where the nucleobases of nucleosides 1 through22 are each complementary to a corresponding position of a miRNA that is23 nucleobases in length, is 100% complementary to a 22 nucleobaseportion of the nucleobase sequence of a miRNA. Such a modifiedoligonucleotide has approximately 96% overall complementarity to thenucleobase sequence of the entire miRNA, and has 100% complementarity toa 22 nucleobase portion of the miRNA.

In certain embodiments, a portion of the nucleobase sequence of amodified oligonucleotide is 100% complementary to a portion of thenucleobase sequence of a miRNA, or a precursor thereof. In certain suchembodiments, 15 contiguous nucleobases of a modified oligonucleotide areeach complementary to 15 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 16 contiguousnucleobases of a modified oligonucleotide are each complementary to 16contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 17 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 17 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 18contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 18 contiguous nucleobases of a miRNA, or a precursorthereof. In certain such embodiments, 19 contiguous nucleobases of amodified oligonucleotide are each complementary to 19 contiguousnucleobases of a miRNA, or a precursor thereof. In certain suchembodiments, 20 contiguous nucleobases of a modified oligonucleotide areeach complementary to 20 contiguous nucleobases of a miRNA, or aprecursor thereof. In certain such embodiments, 22 contiguousnucleobases of a modified oligonucleotide are each complementary to 22contiguous nucleobases of a miRNA, or a precursor thereof. In certainsuch embodiments, 23 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 23 contiguous nucleobases of amiRNA, or a precursor thereof. In certain such embodiments, 24contiguous nucleobases of a modified oligonucleotide are eachcomplementary to 24 contiguous nucleobases of a miRNA, or a precursorthereof.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide consists of 12 to 30linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 15 to 25 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 19 to 24 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 21 to 24linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 12 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 13 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 14 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 15 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 16 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 17 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 18 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 19 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 20 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 21 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 22 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 23 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 24 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 25 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 26 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 27 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 28 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 29 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 30 linkednucleosides. In certain such embodiments, a modified oligonucleotidecomprises linked nucleosides selected from contiguous nucleobases of SEQID NO: 12.

Certain Modifications

Modified oligonucleotides of the present invention comprise one or moremodifications to a nucleobase, sugar, and/or internucleoside linkage. Amodified nucleobase, sugar, and/or internucleoside linkage may beselected over an unmodified form because of desirable properties suchas, for example, enhanced cellular uptake, enhanced affinity for otheroligonucleotides or nucleic acid targets and increased stability in thepresence of nucleases.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more modified nucleosides. In certain suchembodiments, a modified nucleoside is a stabilizing nucleoside. Anexample of a stabilizing nucleoside is a sugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. In certain embodiments, a linked biradical group is selectedfrom —O—, —S—, —N(R₁)—, —C(R₁)(R₂)—, —C(R₁)═C(R₁)—, —C(R₁)═N—,—C(═NR₁)—, —Si(R₁)(R₂)—, —S(═O)₂—, —S(═O)—, —C(═O)— and —C(═S)—; whereeach R₁ and R₂ is, independently, H, hydroxyl, C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀aryl, a heterocycle radical, a substituted heterocycle radical,heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substitutedC₅-C₇ alicyclic radical, halogen, substituted oxy (—O—), amino,substituted amino, azido, carboxyl, substituted carboxyl, acyl,substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O)₂—H),substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; andeach substituent group is, independently, halogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, amino, substituted amino,acyl, substituted acyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ aminoalkoxy,substituted C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkoxy or aprotecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2′and 4′ carbon atoms with a biradical group selected from —O—(CH₂)_(p)—,—O—CH₂CH₂—, —NH—(CH₂)_(p)—, —N(alkyl)-(CH₂)_(p)—, —O—CH(alkyl)-,—(CH(alkyl))-(CH₂)_(p)—, —NH—O—(CH₂)_(p)—, —N(alkyl)-O—(CH₂)_(p)—, or—O—N(alkyl)-(CH₂)_(p)—, wherein p is 1, 2, 3, 4 or 5 and each alkylgroup can be further substituted. In certain embodiments, p is 1, 2 or3.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl;O—, S— or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n))or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-O replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises one or more internucleoside modifications. Incertain such embodiments, each internucleoside linkage of a modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a modified internucleoside linkage comprises a phosphorusatom.

In certain embodiments, a modified oligonucleotide of the presentinvention comprises at least one phosphorothioate internucleosidelinkage. In certain embodiments, each internucleoside linkage of amodified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In certain such embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In certain such embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In certain suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In certain such embodiments, aninternucleoside linkage has an amide backbone. In certain suchembodiments, an internucleoside linkage has mixed N, O, S and CH₂component parts.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified oligonucleotidecomprises one or more 5-methylcytosines. In certain embodiments, eachcytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified nucleobase is selected from5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certainembodiments, a modified nucleobase is selected from 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certainembodiments, a modified nucleobase is selected from 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

Certain Oligonucleotide Motifs

Suitable motifs for modified oligonucleotides of the present inventioninclude, but are not limited to, fully modified, uniformly modified,positionally modified, and gapmer. Modified oligonucleotides having afully modified motif, including a uniformly modified motif, may bedesigned to target mature miRNAs. Alternatively, modifiedoligonucleotides having a fully modified motif, including a uniformlymodified motif, may be designed to target certain sites of pri-miRNAs orpre-miRNAs, to block the processing of miRNA precursors into maturemiRNAs. Modified oligonucleotides having a fully modified motif oruniformly modified motif are effective inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide comprises asugar modification at each nucleoside. In certain such embodiments,pluralities of nucleosides are 2′-O-methoxyethyl nucleosides and theremaining nucleosides are 2′-fluoro nucleosides. In certain suchembodiments, each of a plurality of nucleosides is a 2′-O-methoxyethylnucleoside and each of a plurality of nucleosides is a bicyclicnucleoside. In certain such embodiments, a fully modifiedoligonucleotide further comprises at least one modified internucleosidelinkage. In certain such embodiments, each internucleoside linkage of afully sugar-modified oligonucleotide is a modified internucleosidelinkage. In certain embodiments, a fully sugar-modified oligonucleotidefurther comprises at least one phosphorothioate internucleoside linkage.In certain such embodiments, each internucleoside linkage of a fullysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a fully modified oligonucleotide is modified ateach internucleoside linkage. In certain such embodiments, eachinternucleoside linkage of a fully modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleotide comprisesthe same sugar modification at each nucleoside. In certain suchembodiments, each nucleoside of a modified oligonucleotide comprises a2′-O-methoxyethyl sugar modification. In certain embodiments, eachnucleoside of a modified oligonucleotide comprises a 2′-O-methyl sugarmodification. In certain embodiments, each nucleoside of a modifiedoligonucleotide comprises a 2′-fluoro sugar modification. In certainsuch embodiments, a uniformly modified oligonucleotide further comprisesat least one modified internucleoside linkage. In certain suchembodiments, each internucleoside linkage of a uniformly sugar-modifiedoligonucleotide is a modified internucleoside linkage. In certainembodiments, a uniformly sugar-modified oligonucleotide furthercomprises at least one phosphorothioate internucleoside linkage. Incertain such embodiments, each internucleoside linkage of a uniformlysugar-modified oligonucleotide is a phosphorothioate internucleosidelinkage.

In certain embodiments, a uniformly modified oligonucleotide comprisesthe same internucleoside linkage modifications throughout. In certainsuch embodiments, each internucleoside linkage of a uniformly modifiedoligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises the samesugar modification at each nucleoside, and further comprises one or moreinternucleoside linkage modifications. In certain such embodiments, themodified oligonucleotide comprises one modified internucleoside linkageat the 5′ terminus and one modified internucleoside linkage at the 3′terminus. In certain embodiments, the modified oligonucleotide comprisestwo modified internucleoside linkages at the 5′ terminus and twomodified internucleoside linkages at the 3′ terminus. In certainembodiments, the modified oligonucleotide comprises two modifiedinternucleoside linkages at the 5′ terminus and three modifiedinternucleoside linkages at the 3′ terminus. In certain embodiments, themodified oligonucleotide comprises two modified internucleoside linkagesat the 5′ terminus and four modified internucleoside linkages at the 3′terminus. In certain such embodiments, the modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide is represented by thefollowing formula III:

(5′)QxQz¹(Qy)_(n)Qz²Qz³Qz⁴Q-L(3′)

In certain such embodiments, an compound is represented by formula III.In certain embodiments, Q is a 2′-O-methyl modified nucleoside. Incertain embodiments, x is phosphorothioate. In certain embodiments, y isphosphodiester. In certain embodiments, each of z1, z2, z3, and z4 is,independently phosphorothioate or phosphodiester. In certainembodiments, n is 6 to 17. In certain embodiments, L is cholesterol. Incertain embodiments, n is 12 to 17.

In certain embodiments, x is

One of A and B is S while the other is O;y is

Each of z1, z2, z3, and z4 is independently x or y;n=6-17

L is

Wherein:

X is N(CO)R⁷, or NR⁷;Each of R¹, R³ and R⁹, is independently, H, OH, or —CH₂OR^(b) providedthat at least one of R¹, R³ and R⁹ is OH and at least one of R¹, R³ andR⁹ is —CH₂OR^(b);R⁷ is R^(d) or C₁-C₂₀ alkyl substituted with NR^(c)R^(d) or NHC(O)R^(d);R^(c) is H or C₁-C₆ alkyl;R^(d) is a carbohydrate radical; or a steroid radical, which isoptionally tethered to at least one carbohydrate radical; and

R^(b) is

with one of A and B is S while the other is O.

In certain embodiments, R^(d) is cholesterol. In certain embodimentseach of z¹, z², z³, and z⁴ is

with one of A and B is S while the other is O.

In certain embodiments, R¹ is —CH₂OR^(b). In certain embodiments, R⁹ isOH. In certain embodiments, R¹ and R⁹ are trans. In certain embodiments,R⁹ is OH. In certain embodiments, R¹ and R³ are trans. In certainembodiments, R3 is —CH₂OR^(b). In certain embodiments, R¹ is OH. Incertain embodiments, R¹ and R³ are trans. In certain embodiments, R⁹ isOH. In certain embodiments, R³ and R⁹ are trans. In certain embodiments,R⁹ is CH₂OR^(b). In certain embodiments, R¹ is OH. In certainembodiments, R¹ and R⁹ are trans. In certain embodiments, X is NC(O)R⁷.In certain embodiments, R⁷ is —CH₂(CH₂)₃CH₂NHC(O)R^(d).

In certain embodiments, a positionally modified oligonucleotidecomprises regions of linked nucleosides, where each nucleoside of eachregion comprises the same sugar moiety, and where each nucleoside ofeach region comprises a sugar moiety different from that of an adjacentregion.

In certain embodiments, a positionally modified oligonucleotidecomprises at least 10 2′-fluoro modified nucleosides. Such apositionally modified oligonucleotide may be represented by thefollowing formula I:

5′-T₁-(Nu₁-L₁)_(n1)-(Nu₂-L₂)_(n2)-Nu_(r)(L₃-Nu₃)_(n3)-T₂-3′, wherein:

each Nu₁ and Nu₃ is, independently, a stabilizing nucleoside;

at least 10 Nu₂ are 2′-fluoro nucleosides;

each L₁, L₂ and L₃ is, independently, an internucleoside linkage;

each T₁ and T₂ is, independently, H, a hydroxyl protecting group, anoptionally linked conjugate group or a capping group;

n₁ is from 0 to about 3;

n₂ is from about 14 to about 22;

n₃ is from 0 to about 3; and

provided that if n₁ is 0 then T₁ is not H or a hydroxyl protectinggroup, and if n₃ is 0, then T₂ is not H or a hydroxyl protecting group.

In certain such embodiments, n1 and n3 are each, independently, from 1to about 3. In certain embodiments, n₁ and n₃ are each, independently,from 2 to about 3. In certain embodiments, n₁ is 1 or 2 and n₃ is 2 or3. In certain embodiments, n₁ and n₃ are each 2. In certain embodiments,at least one of n1 and n3 is greater than zero. In certain embodiments,n1 and n3 is each greater than zero. In certain embodiments, one of n1and n3 is greater than zero. In certain embodiments, one of n1 and n3 isgreater than one.

In certain embodiments, n₂ is from 16 to 20. In certain embodiments, n₂is from 17 to 19. In certain embodiments, n₂ is 18. In certainembodiments, n₂ is 19. In certain embodiments, n₂ is 20.

In certain embodiments, about 2 to about 8 of the Nu₂ nucleosides arestabilizing nucleosides. In certain embodiments, from about 2 to about 6of the Nu₂ nucleosides are stabilizing nucleosides. In certainembodiments, from about 3 to about 4 of the Nu₂ nucleosides arestabilizing nucleosides. In certain embodiments, 3 of the Nu₂nucleosides are stabilizing nucleosides.

In certain embodiments, each of the Nu₂ stabilizing nucleosides isseparated from the Nu₃ stabilizing nucleosides by from 2 to about 82′-fluoro nucleosides. In certain embodiments each of the Nu₂stabilizing nucleosides is separated from the Nu₃ stabilizingnucleosides by from 3 to about 8 2′-fluoro nucleosides. In certainembodiments each of the Nu₂ stabilizing nucleosides is separated fromthe Nu₃ stabilizing nucleosides by from 5 to about 8 2′-fluoronucleosides.

In certain embodiments, a modified oligonucleotide comprises from 2 toabout 6 Nu₂ stabilizing nucleosides. In certain embodiments, a modifiedoligonucleotide comprises 3 Nu₂ stabilizing nucleosides.

In certain embodiments, each of the Nu₂ stabilizing nucleosides islinked together in one contiguous sequence. In certain embodiments, atleast two of the Nu₂ stabilizing nucleosides are separated by at leastone of the 2′-fluoro nucleosides. In certain embodiments, each of theNu₂ stabilizing nucleosides is separated by at least one of the2′-fluoro nucleosides.

In certain embodiments, at least two contiguous sequences of the Nu₂2′-fluoro nucleosides are separated by at least one of the stabilizingnucleosides wherein each of the contiguous sequences have the samenumber of 2′-fluoro nucleosides.

In certain embodiments, T₁ and T₂ are each, independently, H or ahydroxyl protecting group. In certain embodiments, at least one of T₁and T₂ is 4,4′-dimethoxytrityl. In certain embodiments, at least one ofT₁ and T₂ is an optionally linked conjugate group. In certainembodiments, at least one of T₁ and T₂ is a capping group. In certainembodiments, the capping group is an inverted deoxy abasic group.

In certain embodiments, a positionally modified oligonucleotidecomprises at least one modified internucleoside linkage. In certain suchembodiments, each internucleoside linkage of a positionally modifiedoligonucleoside is a modified internucleoside linkage. In certainembodiments, at least one internucleoside linkage of a positionallymodified oligonucleotide is a phosphorothioate internucleoside linkage.In certain such embodiments, each internucleoside linkage of apositionally modified oligonucleotide is a phosphorothioateinternucleoside linkage.

In certain embodiments, a positionally modified motif is represented bythe following formula II, which represents a modified oligonucleotideconsisting of linked nucleosides:

T₁(Nu₁)_(n1)-(Nu₂)_(n2)-(Nu₃)_(n3)-(Nu₄)_(n4)-(Nu₅)_(n5)-T₂, wherein:

Nu₁ and Nu₅ are, independently, 2′ stabilizing nucleosides;

Nu₂ and Nu₄ are 2′-fluoro nucleosides;

Nu₃ is a 2′-modified nucleoside;

each of n1 and n5 is, independently, from 0 to 3;

the sum of n2 plus n4 is between 10 and 25;

n3 is from 0 and 5; and

each T₁ and T₂ is, independently, H, a hydroxyl protecting group, anoptionally linked conjugate group or a capping group.

In certain embodiments, the sum of n2 and n4 is 16. In certainembodiments, the sum of n2 and n4 is 17. In certain embodiments, the sumof n2 and n4 is 18. In certain embodiments, n1 is 2; n3 is 2 or 3; andn5 is 2.

In certain embodiments, Nu₁ and Nu₅ are, independently, 2′-modifiednucleosides.

In certain embodiments, Nu₁ is O—(CH₂)₂—OCH₃, Nu₃ is O—(CH₂)₂—OCH₃, Nu₅O—(CH₂)₂—OCH₃, T₁ is H and T₂ is H.

In certain embodiments, a modified oligonucleotide complementary to amiRNA and consisting of 22 linked nucleosides has a Formula II selectedfrom Table A, where each internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, a modifiedoligonucleotide having a Formula II selected from Table A has thenucleobase sequence of SEQ ID NO: 12.

TABLE A n1 n2 n3 n4 n5 Nu₁ Nu₃ Nu₅ T₁ T₂ 2 18 0 0 2 2′- 2′- 2′- H H MOEMOE MOE 2 2 2 14 2 2′- 2′- 2′- H H MOE MOE MOE 2 3 2 13 2 2′- 2′- 2′- HH MOE MOE MOE 2 4 2 12 2 2′- 2′- 2′- H H MOE MOE MOE 2 5 2 11 2 2′- 2′-2′- H H MOE MOE MOE 2 6 2 10 2 2′- 2′- 2′- H H MOE MOE MOE 2 7 2 9 2 2′-2′- 2′- H H MOE MOE MOE 2 8 2 8 2 2′- 2′- 2′- H H MOE MOE MOE 2 9 2 7 22′- 2′- 2′- H H MOE MOE MOE 2 10 2 6 2 2′- 2′- 2′- H H MOE MOE MOE 2 112 5 2 2′- · 2′- 2′- H H MOE MOE MOE 2 12 2 4 2 2′- 2′- 2′- H H MOE MOEMOE 2 13 2 3 2 2′- 2′- 2′- H H MOE MOE MOE 2 14 2 2 2 2′- 2′- 2′- H HMOE MOE MOE 2 2 3 13 2 2′- 2′- 2′- H H MOE MOE MOE 2 3 3 12 2 2′- 2′-2′- H H MOE MOE MOE 2 4 3 11 2 2′- 2′- 2′- H H MOE MOE MOE 2 5 3 10 22′- 2′- 2′- H H MOE MOE MOE 2 6 3 9 2 2′- 2′- 2′- H H MOE MOE MOE 2 7 38 2 2′- 2′- 2′- H H MOE MOE MOE 2 8 3 7 2 2′- 2′- 2′- H H MOE MOE MOE 29 3 6 2 2′- 2′- 2′- H H MOE MOE MOE 2 10 3 5 2 2′- 2′- 2′- H H MOE MOEMOE 2 11 3 4 2 2′- 2′- 2′- H H MOE MOE MOE 2 12 3 3 2 2′- 2′- 2′- H HMOE MOE MOE 2 13 3 2 2 2′- 2′- 2′- H H MOE MOE MOE 2 8 6 4 2 2′- 2′- 2′-H H MOE MOE MOE

A modified oligonucleotide having a gapmer motif may have an internalregion consisting of linked 2′-deoxynucleotides, and external regionsconsisting of linked 2′-modified nucleosides. Such a gapmer may bedesigned to elicit RNase H cleavage of a miRNA precursor. The internal2′-deoxynucleoside region serves as a substrate for RNase H, allowingthe cleavage of the miRNA precursor to which a modified oligonucleotideis targeted. In certain embodiments, each nucleoside of each externalregion comprises the same 2′-modified nucleoside. In certainembodiments, one external region is uniformly comprised of a first2′-modified nucleoside and the other external region is uniformlycomprised of a second 2′-modified nucleoside.

A modified oligonucleotide having a gapmer motif may have a sugarmodification at each nucleoside. In certain embodiments, the internalregion is uniformly comprised of a first 2′-modified nucleoside and eachof the wings is uniformly comprised of a second 2′-modified nucleoside.In certain such embodiments, the internal region is uniformly comprisedof 2′-fluoro nucleosides and each external region is uniformly comprisedof 2′-O-methoxyethyl nucleosides.

In certain embodiments, each external region of a gapmer consists oflinked 2′-O-methoxyethyl nucleosides. In certain embodiments, eachexternal region of a gapmer consists of linked 2′-O-methyl nucleosides.In certain embodiments, each external region of a gapmer consists of2′-fluoro nucleosides. In certain embodiments, each external region of agapmer consists of linked bicyclic nucleosides.

In certain embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises a different 2′-modification. Incertain such embodiments, each nucleoside of one external region of agapmer comprises 2′-O-methoxyethyl nucleosides and each nucleoside ofthe other external region comprises 2′-O-methyl nucleosides. In certainsuch embodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises 2′-fluoro nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methoxyethyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides. In certain suchembodiments, each nucleoside of one external region of a gapmercomprises 2′-O-methyl nucleosides and each nucleoside of the otherexternal region comprises bicyclic nucleosides.

In certain embodiments, nucleosides of one external region comprise twoor more sugar modifications. In certain embodiments, nucleosides of eachexternal region comprise two or more sugar modifications. In certainembodiments, at least one nucleoside of an external region comprises a2′-O-methoxyethyl sugar and at least one nucleoside of the same externalregion comprises a 2′-fluoro sugar. In certain embodiments, at least onenucleoside of an external region comprises a 2′-O-methoxyethyl sugar andat least one nucleoside of the same external region comprises a bicyclicsugar moiety. In certain embodiments, at least one nucleoside of anexternal region comprises a 2′-O-methyl sugar and at least onenucleoside of the same external region comprises a bicyclic sugarmoiety. In certain embodiments at least one nucleoside of an externalregion comprises a 2′-O-methyl sugar and at least one nucleoside of thesame external region comprises a 2′-fluoro sugar. In certainembodiments, at least one nucleoside of an external region comprises a2′-fluoro sugar and at least one nucleoside of the same external regioncomprises a bicyclic sugar moiety.

In certain embodiments, each external region of a gapmer consists of thesame number of linked nucleosides. In certain embodiments, one externalregion of a gapmer consists a number of linked nucleosides differentthat that of the other external region.

In certain embodiments, the external regions comprise, independently,from 1 to 6 nucleosides. In certain embodiments, an external regioncomprises 1 nucleoside. In certain embodiments, an external regioncomprises 2 nucleosides. In certain embodiments, an external regioncomprises 3 nucleosides. In certain embodiments, an external regioncomprises 4 nucleosides. In certain embodiments, an external regioncomprises 5 nucleosides. In certain embodiments, an external regioncomprises 6 nucleosides. In certain embodiments, the internal regionconsists of 17 to 28 linked nucleosides. In certain embodiments, aninternal region consists of 17 to 21 linked nucleosides. In certainembodiments, an internal region consists of 17 linked nucleosides. Incertain embodiments, an internal region consists of 18 linkednucleosides. In certain embodiments, an internal region consists of 19linked nucleosides. In certain embodiments, an internal region consistsof 20 linked nucleosides. In certain embodiments, an internal regionconsists of 21 linked nucleosides. In certain embodiments, an internalregion consists of 22 linked nucleosides. In certain embodiments, aninternal region consists of 23 linked nucleosides. In certainembodiments, an internal region consists of 24 linked nucleosides. Incertain embodiments, an internal region consists of 25 linkednucleosides. In certain embodiments, an internal region consists of 26linked nucleosides. In certain embodiments, an internal region consistsof 27 linked nucleosides. In certain embodiments, an internal regionconsists of 28 linked nucleosides.

Certain Quantitation Assays

The effects of antisense inhibition of a miRNA following theadministration of modified oligonucleotides may be assessed by a varietyof methods known in the art. In certain embodiments, these methods arebe used to quantitate miRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in miRNA levels are measured bymicroarray analysis. In certain embodiments, changes in miRNA levels aremeasured by one of several commercially available PCR assays, such asthe TaqMan® MicroRNA Assay (Applied Biosystems). In certain embodiments,antisense inhibition of a miRNA is assessed by measuring the mRNA and/orprotein level of a target of a miRNA. Antisense inhibition of a miRNAgenerally results in the increase in the level of mRNA and/or protein ofa target of the miRNA.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. In certain embodiments, experimental models areemployed to evaluate the effectiveness of modified oligonucleotides ofthe invention for the treatment of fibrosis. Those having skill in theart are able to select and modify the protocols for such experimentalmodels to evaluate a pharmaceutical agent of the invention.

Modified oligonucleotides may first be tested in cultured cells.Suitable cell types include those that are related to the cell type towhich delivery of a modified oligonucleotide is desired in vivo. Forexample, suitable cell types for the study of modified oligonucleotidesfor the treatment of fibrosis include fibroblasts, cardiomyocytes, andstellate cells.

In certain embodiments, the extent to which a modified oligonucleotideinhibits the activity of a miRNA is assessed in cultured cells. Incertain embodiments, inhibition of miRNA activity may be assessed bymeasuring the levels of the miRNA. Alternatively, the level of apredicted or validated miRNA target may be measured. An inhibition ofmiRNA activity may result in the increase in the mRNA and/or protein ofa miRNA target. Further, in certain embodiments, certain phenotypicoutcomes may be measured. For example, suitable phenotypic outcomesinclude inhibition of cell proliferation, the induction of cell death,and/or the induction of apoptosis.

Following the in vitro identification of a modified oligonucleotide thateffectively inhibits the activity of a miRNA, modified oligonucleotidesare further tested in in vivo experimental models.

Suitable experimental models for the testing of pharmaceutical agentsfor the treatment of fibrosis, including pharmaceutical agentscomprising modified oligonucleotides complementary to a miR-122, includea pressure overload-induced hypertrophy model, described herein.

An additional experimental model for the testing of pharmaceuticalagents for the treatment of fibrosis includes, but is not limited to,the methionine choline deficient (MCD) diet model (see, for example,Yamaguchi et al., Hepatology, 2008, 47, 625-635). db/db micespontaneously develop obesity, diabetes, and fatty livers. Feeding suchmice a MCD diet induces non-alcoholic steatohepatitis (NASH) and liverfibrosis within 4 to 8 weeks. Modified oligonucleotides havingnucleobase complementary to a miRNA are tested in this model for theireffects on liver fibrosis.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES Example 1: A Role for miR-21 in Cardiac Disease

Microarray analysis in a transgenic mouse model of cardiac failure(heart-restricted overexpression of the β1-adrenergic receptor(Engelhardt et al., 1999)) revealed progressive de-regulation of thecardiac microRNA expression signature with increasing severity of thedisease (FIG. 1 a). While miR-21 expression was modest in normalmyocardium, this microRNA was among the strongest regulated microRNAs inthe failing heart. In fact, in mice with end-stage heart failure, miR-21was the single strongest upregulated microRNA (FIG. 1 a). QuantitativeNorthern blot analysis confirmed upregulation of miR-21 in the mouse andfurther revealed a marked upregulation in human heart failure (FIG. 1 b,c). Increased expression of the miR-21 precursor as assessed by Northernblotting suggested a transcriptional mechanism. Thus, the human miR-21promoter was studied in more detail. A miR-21 promoter region wasidentified and is highly conserved in several species (FIG. 4 a).Studies on human miR-21 expression (FIG. 4 a-c) confirmed itstranscriptional regulation by two transcription factors, calcium/cAMPresponse element protein (CREB) and serum response factor (SRF), whichare classically activated during the cardiac stress response. Deletionof CREB and mutation of the SRF binding sites in the miR-21 promoterresulted in markedly decreased miR-21 expression in response to serumstimulation, indicating a major role for these two transcription factorsin miR-21 regulation (FIG. 4 c). An essential role for miR-21 in cardiacmorphology and function was detected by a morpholino-based approach in abiased screen to knock down several ubiquitously expressed microRNAs inzebrafish. Knockdown of miR-21 led to a dramatic impairment of cardiacstructure and function (FIG. 5). More than 95% of injected animalsdisplayed massive pericardial effusions (FIG. 5 a, inset and b) andimpaired ventricular function as determined by video microscopy (FIG. 5c).

To study these essential functions of miR-21 in the mammalian heart infurther detail, the inventors modulated miR-21 expression in isolatedcardiomyocytes. The inventors transfected synthetic miR-21-precursors aswell as antisense miR-21-inhibitors, routinely achieving >95%transfection efficiency of oligonucleotide delivery (FIG. 6 a).Overexpression of miR-21 led to a robust enhancement of mature miR-21,while inhibition of miR-21 completely suppressed endogenous miR-21expression as determined by Northern blot analysis (FIG. 6 b). However,neither enhancement nor suppression of miR-21 levels in cardiomyocytessignificantly affected the morphology, size or number of primary ratcardiomyocytes under resting conditions or under conditions ofcardiomyocyte hypertrophy (FIG. 1 d). Cardiomyocyte-specific miR-21transgenic mice overexpressing miR-21 25-fold relative to wildtypelittermates displayed no obvious cardiac phenotype with intact structureof the left ventricular myocardium and absence of interstitial fibrosis(FIG. 1 e, bottom). Contrary to the substantially increased miR-21expression in the failing heart, these data show that manipulation ofmiR-21 expression levels in isolated cardiomyocytes andcardiomyocyte-specific miR-21 transgenic mice models fails tocorroborate a significant function of miR-21 in the heart as indicatedby the observed effects in zebrafish and the substantially increasedmiR-21 expression in the failing heart. Therefore, the inventors nextexplored a potential role of miR-21 in a non-cardiomyocyte cell type,e.g. cardiac fibroblasts. Using in situ hybridization, weak miR-21signals were detected in normal myocardium, whereas in failingmyocardium the hybridization signal was greatly enhanced. At highmagnification, the hybridization signal was restricted primarily tosmall interstitial cells, presumably cardiac fibroblasts. Using apre-plating procedure, the inventors fractionated neonatal rat heartsinto a cardiomyocyte and fibroblast fraction. Indeed, the inventorsdetected endogenous miR-21 expression primarily in cardiac fibroblasts(FIG. 1 f).

Example 2: miR-21 Derepresses Cardiac Fibroblast ERK-Signaling

MiR-21 expression is selectively increased in various human cancers (Luet al., 2005; Iorio et al., 2005) and was shown to contribute to tumorgrowth and spread by diverse mechanisms in different cells. Since miR-21appears to target distinct mRNAs in a cell type-specific manner, theinventors decided to investigate potential heart specific miR-21targets. Using a bioinformatic approach the inventors screened severalmicroRNA databases for potential miR-21 targets with 3′ UTR-comprisingseed sequences and matching flanking nucleotides and focused thisanalysis on candidates with reported cardiac expression. A summary isdepicted in Table 1 below.

TABLE 1 cardiac number of expression conserved Predicted Predicted Seedmatch gene (ID species target target for miR-21 symbol gene nameReference) (miRBase) (miRBase) (PicTar) (Target Scan) Spry1 sprouty15306693 7 yes yes 8mer homolog 1 Tgfbi transforming 10913330 7 yes yes8mer growth factor, beta induced Krit1 KRIT1, 16455310 7 yes yes 7merankyrin repeat containing Pitx2 paired-like 16836994 7 yes yes 7merhomeodomain transcription factor 2 Fasl Fas ligand 16698421 7 yes yes7mer (TNF superfamily, member 6) Nfib nuclear facor 9056636 7 yes no7mer IB Lnx1 ligand of 17118964 7 yes no 7mer numb-protein X1 Rtn4reticulon 4 16202479 8 yes no 7mer

A screen for theoretical miR-21 targets revealed 22 known potentialtarget genes, of which 8 were previously shown to be expressed withincardiac tissue. Combination of three different target prediction toolsidentified Spry1 (sprouty1) as a highly likely candidate. Sprouty1(SPRY1), a known inhibitor of the Ras/MEK/ERK-pathway (Hanafusa et al.,2002, Casci et al., 1999) emerged as a potential target due to its highscore and significant expression level in the heart (Table 1). The 3′UTRof the Spry1 mRNA contains several predicted microRNA binding sites, ofwhich only one corresponds to a microRNA highly upregulated duringcardiac disease (miR-21, see FIG. 7). To determine the cell type inwhich Spry1 is expressed, the inventors employed an allele of Spry1, inwhich the lacZ gene replaces part of the Spry1 coding sequence. Assaysfor lacZ expression showed prominent staining in the adult mouse heart(FIG. 2 a). Higher magnification identified a spotted pattern of Spry1expression originating from interstitial fibroblasts (FIG. 2 a, lowerpanel). Thus, both miR-21 and its putative target, Spry1, areco-expressed in cardiac fibroblasts, and not in the cardiomyocytefraction. In accordance with these observations, no detectabledownregulation of SPRY1 expression was found in transgenic miceoverexpressing miR-21 in a cardiomyocyte-specific manner. In addition,cardiac CREB, the transcriptional activator of miR-21 expression (FIG.4) is localized exclusively in fibroblasts. The inventors then testedthe relevance of these findings to human disease. Indeed, analysis ofleft ventricular cardiac tissue samples from patients with end-stageheart failure due to idiopathic dilated cardiomyopathy demonstratedincreased miR-21 expression (FIG. 1c ) and significant repression ofSPRY1 protein expression (FIG. 2 b). These findings were accompanied byactivation of ERK-MAPkinase, as evidenced by an increasedphospho-ERK/ERK ratio (FIG. 2 b).

MiR-21 functions were then characterized in co-cultures of interstitialfibroblasts and cardiomyocytes to mimic the composition of intactcardiac tissue. The inventors assessed miR-21 function by transfectionof synthetic miR-21 precursor molecules or inhibitors. Increasing miR-21induced a strong repression of SPRY1 protein expression and increasedERK-MAPkinase activation (FIG. 2 c). SiRNA-mediated Spry1 silencinglikewise resulted in ERK-MAPkinase activation (FIG. 2 d). The inventorsnext evaluated whether miR-21-mediated derepression of fibroblastERK-MAPkinase signaling would suffice to affect fibroblast survival. Inagreement with a potential role of ERK-MAPkinase signaling in myocardialfibrosis, enhancement of miR-21 levels promoted cardiac fibroblastsurvival, whereas suppression of endogenous miR-21 induced apoptoticcell death (FIG. 2 e). The inventors also found miR-21-based regulationof SPRY1 expression to be critical for the secretory function of cardiacfibroblasts, as both overexpression of miR-21 as well as siRNA-mediatedsilencing of SPRY1 expression significantly augmented secretion offibroblast growth factor 2 (FGF2) into the supernatant (FIG. 2 f). Thisstudy thus delineated a novel signaling paradigm in the failing heart,where re-expression of miR-21 during cardiac disease augmentsERK-MAPkinase activity through inhibition of SPRY1. In the mammalianheart, this mechanism may regulate fibroblast survival and therebycritically govern the extent of interstitial fibrosis and cardiacremodeling.

Example 3: Therapeutic Silencing of miR-21 In Vivo

To evaluate the function of miR-21 in vivo, in a normal setting and inheart failure, miR-21 activity was inhibited using modifiedoligonucleotide complementary to miR-21 (miR-21 antagonist). A pressureoverload-induced hypertrophy mouse model in was used as a model forhuman heart failure. In this model a reproducible cardiac stressresponse is achieved through transverse aortic constriction (TAC). Thismodel highly resembles the failing human heart due to its matchingpattern of both microRNA and mRNA changes in global expression profiles(Thum et al. 2007).

To determine the localization of antagomir-21 within the heart, aCy-3-labeled antagomir-21 was injected intravenously via a jugular veincatheter. Strong staining throughout the left ventricular myocardium wasobserved (FIG. 3 a), indicating that modified oligonucleotidescomplementary to miR-21 achieve distribution to cardiac tissue. ThemiR-21 antagonist comprised 2′-O-methyl sugars at each nucleoside, twophosphorothioate internucleoside linkages at the 5′-most end of theoligonucleotide, three phosphorothioate internucleoside linkages at the3′-most end of the oligonucleotide, and a cholesterol linked through ahydroxyprolinol linker. miR-21 antagonists had the nucleobase sequenceof SEQ ID NO: 1 or SEQ ID NO: 12. Untreated (sham) or TAC-operated micewere treated with antagomir-21 or a control oligonucleotide at doses of80 mg/kg for three consecutive days. Treatment with antagomir-21strongly repressed elevated cardiac miR-21 expression for up to threeweeks as determined in Northern blots (FIG. 3 b) and real-time PCRanalysis (FIG. 8). This treatment completely reversed TAC-induceddownregulation of SPRY1 and ERK-MAPkinase activation during pressureoverload to levels observed in sham-operated mice (FIG. 3 c).Interstitial fibrosis and heart weight were significantly increasedthree weeks after TAC in untreated mice, but were strongly attenuated byantagomir-21 treatment (FIGS. 3 d, e). Indeed, collagen content of themyocardium was essentially normalized by antagomir-21 treatment.Further, where heart weight doubled three weeks after TAC, antagomir-21treatment prevented hypertrophy. In sham-operated mice, treatment withantagomir-21 did not result in significant changes, in cardiac weight orinterstitial fibrosis, indicating no discernable effect of miR-21antagonism on the normal heart or overt cardiotoxicity of antagomir-21treatment. After TAC operation and antagomir-21 treatment, globaltranscriptome analysis revealed normalization of various deregulatedgenes (FIG. 3 f). Specifically, genes highly upregulated during cardiacfibrosis such as collagen 1α1, collagen 3α1, biglycan, fibromodulin orconnective tissue growth factor were reduced after specific inhibitionof miR-21 by 42%, 39%, 44%, 38% and 37%, respectively (FIG. 3 f lowerpanel). In further studies, cardiac function was assessed byechocardiography. Left ventricular end-diastolic diameters increasedsignificantly three weeks after TAC and fractional shortening wasimpaired (FIG. 3 g), as is commonly observed in human heart failure.When compared with controls, antagomir-21 treatment prevented leftventricular dilatation and normalized parameters of fractionalshortening essentially to levels observed in sham-operated animals (FIG.3 g). Similar results were obtained with antagomir-21 treatment in anisoproterenol-induced cardiac disease model.

An additional experiment was performed, in which mice were subject topressure overload of the left ventricle for three weeks before treatmentwith antagomir-21. During this period the animals displayed significantleft ventricular hypertrophy, fibrosis and impaired cardiac function.After this three week time period, mice were treated with antagomir-21and observed for an additional three weeks. Whereas animals treated withcontrol displayed progressive impairment of left ventricular function aswell as interstitial fibrosis and cardiac hypertrophy, animals treatedwith antagomir-21 showed significant attenuation of the impairment ofcardiac function as well as regression of cardiac hypertrophy andfibrosis.

These data demonstrate a critical role for fibroblast-derived miR-21 andSPRY1 in the heart. Abnormal expression of miR-21 in cardiac fibroblastsinhibits SPRY1 protein expression, resulting in augmentation of ERK-MAPkinase activity. In turn, this enhances cardiac fibroblast survival andthereby the interstitial fibrosis and cardiac remodeling that ischaracteristic of the failing heart. This model (summarized in FIG. 3 h)assigns a primary role to cardiac fibroblast activation in myocardialdisease. Antagonizing miR-21 in the murine model of heart diseaseprevented structural and function deterioration. Accordingly, thepresent invention provides methods for the treatment of fibrosis,comprising administering a modified oligonucleotide complementary tomiR-21. The present invention further provides methods for the treatmentof fibrosis related to cardiac disease, comprising administering amodified oligonucleotide complementary to miR-21.

Example 4: miR-21 Regulation of the Extracellular Signal-RegulatedKinase MAPK Pathway

Malignant transformation of normal to cancer cells requires theacquisition of several oncogenic traits such as uncontrolled celldivision, resistance to programmed cell death (apoptosis), invasion andangiogenesis. Genetic alterations often result in constitutiveactivation of common downstream signal transduction pathways such as themitogen activated protein (MAP) kinase cascade involving c-RAF-1, MEK-1,ERK 1/2, p38 and JNK and others. Mitogen-activated protein kinase (MAPK)cascades are key signaling pathways involved in the regulation of normalcell proliferation, survival and differentiation. Aberrant regulation ofMAPK cascades contribute to cancer and other human diseases. Inparticular, the extracellular signal-regulated kinase (ERK) MAPK pathwayhas been the subject of intense research leading to the development ofpharmacologic inhibitors for the treatment of cancer. In normal cells,it is well established that the activation of the kinase ERK 1/2 isregulated by receptor tyrosine kinases such as EGF-receptors andplatelet-derived growth factor receptors (PDGFRs) via activation of Ras,which in turn activates the RAF-1/MEK-1/ERK 1/2 cascade. Because thissignal transduction pathway is hyperactivated in many human cancers,inhibitors of receptor tyrosine kinases, Ras, c-RAF-1 and MEK-1 have allbeen developed and are at various stages of development. The treatmentof cancer may therefore be possible by administering an effective amountof an inhibitor of the RAF-1/MEK-I/P-ERK 1/2 pathway.

Sprouty (SPRY) is a family of intracellular proteins that are endogenousregulators of receptor tyrosine kinase pathways such as the Ras/MAPkinase pathway. Mammalian species express four isoforms of Sprouty,which act as inhibitors of growth factor-induced cellulardifferentiation, migration, and proliferation. The inventors identifiedsprouty-1 to inhibit ERK phosphorylation which may result in modulationof tumor formation. The inventors suggest that upregulation of miR-21 inmany human cancers inhibits sprouty-1 thus activating ERK to result inenhanced tumor formation and progression. Antagonism of miR-21 (e.g. byan antagomir-21) is therefore able to prevent and/or attenuate tumorformation and/or progression.

Example 5: Experimental Procedures Expression Analyses (MicroRNA Array,Affymetrix Genechip Analysis, Northern Blotting, Real-Time PCR)

MicroRNA (Castoldi et al., 2007) and global mRNA expression profileswere generated from RNA preparations from murine left ventricularmyocardium. Deregulated microRNAs were confirmed by Northern blottingand stem-loop specific real-time PCR. For both oligonucleotide arraysand spotted microRNA arrays, data analysis was done with the use of Rpackages from the Bioconductor project (www.bioconductor.org) aspreviously described (Thum et al., 2007).

miR-21 Promoter Analysis

24 hours after isolation, cells were transfected with 1 ug of reporterplasmid using an established liposomal transfection method(Lipofectamine, Invitrogen, USA). After 12 hours medium was changed toFCS-free medium. 24 hours later cells were treated either with 5% FCSfor 8 hours whereas the other group was cultured without FCS. Luciferaseactivity was measured in cell lysates using the Dual Luciferase Kit(Promega, Germany) according to the manufacturer's recommendation.

Cardiomyocyte Isolation, Culture and Transfection Experiments

Neonatal cardiomyocytes were isolated as described previously (Merkle etal., 2007). Cardiomyocyte size was determined from digitally recordedimages using the AxioVision LE 4.1 software package (Carl Zeiss VisionGmbH, Jena, Germany). Cardiomyocyte cultures were transfected withprecursor and inhibitors of miR-21 (Ambion, USA) or siRNA againstsprouty1 (Promega, Germany). Using appropriate culture conditions theinventors also performed experiments with cardiac fibroblasts andfibroblast/cardiomyocyte co-cultures.

Zebrafish Maintenance, Microinjection of Morpholino AntisenseOligonucleotides and Determination of Cardiac Function

A standard morpholino-modified oligonucleotide was directed against themature dre-miR-21 (MO-1=5′-GCCAACACCAGTCTGATAAGCTA-3′) and also amulti-blocking Morpholino-modified oligonucleotide was used to interferewith multiple steps in miR-21 processing and function(MO-2=5″-TGTAACAGCCAACACCAGTCTGATAAGCTAT-3′). A standard controloligonucleotide (MO-control) (GENETOOLS, LLC) was injected at the sameconcentration as a negative control. Morpholinos were microinjected intoone-cell stage wild-type zebrafish embryos and overall morphology andespecially heart function was evaluated at several time points duringdevelopment. Pictures and movies were recorded and ventricularfractional shortening was measured 48, 72, 80, 96 and 120 hours postfertilization (hpf) essentially as described (Rottbauer et al., 2005).

Mouse Models of Cardiac Hypertrophy and Failure

Transaortic constriction was performed by routine methods.Beta1-adrenergic receptor transgenic mice (line TG4) have been describedin detail previously (Engelhardt et al., 1999).

Human Heart Samples

The inventors examined cardiac tissue from patients undergoing hearttransplantation because of end-stage heart failure due to dilatativecardiomyopathy and, for comparison, healthy adult heart samples.Immediately after explantation, tissue pieces were removed from the leftventricles, and excised tissue was shockfrozen in liquid nitrogen andstored at −80° C. until analysis.

microRNA Target Prediction Methods

The microRNA databases and target prediction tools miRBase(http://microrna.sanger.ac.uk/), PicTar (http://pictar.bio.nyu.edu/) andTargetScan (http://www.targetscan.org/index.html) were used to identifypotential microRNA targets.

Western Blotting and Analysis of Cardiac Fibrosis

Protein lysates from explanted hearts or co-cultures were prepared asdescribed (Buitrago et al., 2005) and expression of Spry1, ERK1/2,phosphoERK1/2 and G beta detected as described herein. For analysis ofmorphology and fibrosis hearts were fixed in 4% formalin and embedded inparaffin. Tissue sections (5 um) from the LV were stained withhematoxylin and eosin or picrosirius red. Picrosirius red sections wereexamined using a Nikon ECLIPSE 50i microscope equipped with filters toprovide circularly polarized illumination. Tissue images were obtainedwith a 20× objective lens, recorded on a cooled digital camera (DS-5Mc,Nikon), and analyzed using SigmaScan Pro 5.0 image analysis software(SPSS Inc., USA). Collagen content was calculated as a percentage of thearea of each image (expressed in pixels).

αMHC-miR-21 Transgenic Mice

Transgenic mice overexpressing miR-21 were generated by pronuclearinjection of fertilized oocytes from FVB/N mice with a transgeneconstruct containing the mature miR-21 sequence, flanked by 154 bpupstream and 136 bp downstream of the native precursor sequence underthe control of the murine α-myosin heavy chain (αMHC) promoter.

X-Gal Staining of Myocardium from Spry-lacZ Mice

Hearts were collected from Spry1-lacZ+/−mice and fixed for 2 h in PBScontaining 2% formaldehyde and 0.05% glutaraldehyde. Subsequently, thehearts were rinsed 4 times for 30 min in 0.01% Na-desoxycholate, 0.02%Nonidet P-40, 2 mM MgCl2 and 2 mM EGTA in PBS. For the detection of theβ-galactosidase activity the hearts were incubated in rinsing solutioncontaining 0.5 mg/ml X-gal, 10 mM K3Fe(CN)6, and 10 mM, K4Fe(CN)6 at 37°C. For whole mount analysis the hearts were transferred to 30% glyceroland digital images were taken using a Nikon Digi-tal Camera DXM1200F andACT-1 software. For histological analysis the hearts were dehydrated inisopropanol, cleared in xylene and transferred to paraffin. 10 urnparaffin sections were generated using standard protocols anddocumented.

Injection and Detection of Modified Oligonucleotides

A jugular vein catheter was permanently inserted in male 057/B16 mice(10-12 weeks old) before TAC procedure. 24 h post-TAC 80 mg/kg/d ofmodified oligonucleotide was injected daily for 3 days through thejugular vein catheter. As a positive control for effective delivery(Cy3-labeled modified oligonucleotide) was injected at 80 mg/kg onceinto the catheter and the heart was removed 3 h later, fixed and Cy3staining observed by fluorescence microscopy.

Fibroblast Apoptosis and FGF2 Production

After treatment with miR-21 precursors, inhibitors or respectivecontrols, Annexin V-positive fibroblasts were measured by FACS analysis(Annexin-VFLUOS kit, Roche Diagnostics GmbH, Mannheim, Germany).Enzyme-linked immunosorbent assay (ELISA) was performed to quantify FGF2concentrations in supernatants of miR-21 modulated cardiac fibroblasts.The FGF2 assay was carried out using the Quantikine FGF BasicImmunoassay kit (R&D Systems, Minneapolis, USA) according to themanufacturer's specifications.

Statistical Analysis

Average data are presented as mean±SEM. Statistical analysis was carriedout using the Prism software (GraphPad, San Diego, Calif.) or StatView(SAS Institute Inc., Cary, USA) package. ANOVA followed by Bonferroni'stest and Student's t-test were used as appropriate. Differences wereconsidered significant when P<0.05 and are indicated by an asterisk. *indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005.

RNA Isolation, Real-Time RT-PCR and Northern Blotting

For extraction of total RNA from frozen tissues or cell cultures theRNeasy Mini Kit (Qiagen, Hilden, Germany) was used according to themanufacturer's instructions. MiRNAs were isolated by TRIZOL (Invitrogen,Karlsruhe, Germany) or a miRNA Isolation Kit (mirVana™, Ambion, USA).The integrity of the isolated RNA was verified with denaturing agarosegel electrophoresis or capillary electrophoresis (Bioanalyzer 2100;Agilent) as described (Thum and Borlak, 2004). For real-time PCR theinventors employed an iCycler IQ™ Real-Time PCR Detection System(BioRad, Germany).

The inventors used target-specific stem loop structure and reversetranscription primer, and after reverse transcription used specificTaqMan hybridization probes to quantify miR-21 expression (TaqMan miR-21MicroRNA Assay, Applied Biosystems, Foster City, USA). The small RNAmolecule U6B small nuclear (RNU6B) was amplified as a control (TaqManMicroRNA Assay Controls, Applied Biosystems, Foster City, USA). AllmiRNA samples were derived from isolations comprising the same total RNAconcentration.

For Northern blot analysis, 3 μg of total RNA were loaded onto 15%acrylamide, 6 M urea, and TBE gels alongside an appropriate DNA marker.Following electrophoresis, RNA was transferred to a nylon membrane(Qiabrane Nylon, Qiagen) using semidry transfer. Membranes were thenpre-hybridized for 1 h at 65° C. in hybridization buffer (ULTRAhyb-OligoHybridzation Buffer, Ambion, USA). LNA oligonucleotides (miRCURY LNAArray detection probes; Exiqon) that were previously labeled with T4kinase (Exiqon) and 32P-ATP were then added to the buffer and membraneswere hybridized over night at 42° C. Subsequently, the blot was washedthree times at room temperature for 3 minutes (in 0.2×SSC), followed byone time at 42° C. for 15 minutes. Afterwards membranes were exposed ona phosphoimager.

microRNA Expression Analyses

For microarray microRNA expression analyses the inventors separatelypurified microRNAs using the flashPAGE Fractionator system (Ambion,USA). microRNA obtained from 8 μg total RNA was labeled with the dye Cy3(Molecular Probes, Carlsbad, Calif.) by use of the mirVana microRNALabeling Kit (Ambion, USA) according to the manufacturer'srecommendations. Each sample was hybridized to a separate array.MicroRNA microarray hybridization, microRNA purification and enrichment,labeling and microarray hybridization procedures were performedaccording to the Ambion mirVana manuals (www.ambion.com/techlib/prot/)or as described 2. Data acquisition was done with the use of ScanAlyzeSoftware (Eisen-Lab, Lawrence Berkeley National Lab (LBNL), Berkeley,USA). Alternatively, 5 μg total RNA was labeled with a Cy3-conjugatedRNA linker (Dharmacon, USA) and hybridized to a microarray platform forgenome-wide profiling of miRNAs [miChip; capture probes immobilized onthe array correspond to (211 human, 51 murine) unique human miRNAs asdeposited in miRbase version 6.1]. Hybridization signal intensities wereacquired using the Axon scanner (4000B, Molecular Dynamics) withidentical photomultiplier settings. Further analyses were performedusing Genepix 6 (Molecular Dynamics) and Excel softwares. MiR-21expression was validated by specific TaqMan RT-PCR analyses and NorthernBlot analysis.

Global Transcriptome Analysis

For transcriptome analyses, reverse transcription, second-strandsynthesis, and cleanup of double-stranded cDNA were performed accordingto the Affymetrix protocols (One-Cycle cDNA synthesis Kit, Affymetrix,USA) starting from 2 μg of total RNA (n=4 control hearts aftersham-surgery, n=4 left ventricles after TAO and placebo treatment; n=4left ventricles after TAC and miR-21 antagonist treatment). Synthesis ofbiotin-labeled cRNA was performed with the use of the IVT Labeling Kit(Affymetrix, USA). cRNA concentration was determined and thedistribution of cRNA fragment sizes was checked by gel electrophoresis.15 μg of fragmented cRNA was used for hybridization on the mouse genome430 2.0 GeneChip (Affymetrix, USA). Data analysis from array studies Rpackages from the Bioconductor project (www.bioconductor.org) were used.Resulting signal intensities were normalized by variance stabilization.Quality of all data sets was tested and statistical analysis wasperformed using the limma (Linear Models for Microarray Analysis)package to select for differentially expressed genes.

Cardiac Co-Culture Experiments and microRNA/siRNA TransfectionProcedures

Cardiomyocytes from neonatal rats were isolated and cultivated using asdescribed (Merckle et al., 2007). For analysis of miR-21 modulation oncardiomyocyte size, pure cardiomyocyte cultures were used by addition ofpre-plating steps to exclude major non-cardiomyocyte (e.g. fibroblasts)contaminations. More than 95% of cultured cardiomyocytes stainedpositive for actinin, demonstrating high purity of cell cultures.Scrambled-miR (prenegative control #2, Ambion; 50 nmol/L, 72 hours),miR-21 precursor molecules (pre-MiR, Ambion; 50 nmol/L, 72 hours) ormiR-21 antagonists (anti-miR, Ambion; 50 nmol/L, 72 hours) weretransfected by a liposomal-based method (Lipofectamine, Invitrogen, USA;see 6 for details). Cardiomyocytes were cultured with low FCS (0.1%,control condition) or 48 h with high FCS (5.0%) to induce cardiomyocytehypertrophy. For determination of cell size, surface area of neonatalcardiomyocytes (72 hours after transfection) was calculated in a 96-wellplate format (seeding density 40.000 cells/well) with the use of theAxioVison Rel 4.4 package (Carl Zeiss GmbH, Jena, Germany). Data areexpressed as mean±SEM.

To mimic cardiac in vivo conditions the inventors used a co-culturesystem of cardiomyocytes and cardiac fibroblasts by ignoring thepreplating step as described above. Here, the inventors studied indetail expression of Spry-1 and Erk activation after transfection ofscrambled-miR (50 nmol/L, 72 hours), miR-21 precursor molecules (50nmol/L, 72 hours) or miR-21 antagonists (50 nmol/L, 72 hours). Inseparate experiments scrambled siRNA or a specific siRNA cocktailagainst Spry1 (three different microRNAs, 16.7 nmol/L each) weretransfected to the co-culture. Transfection efficiency was monitored byrealtime-PCR measurements (TaqMan MicroRNA Assays, Applied Biosystems)and Northern blotting.

microRNA Target Prediction Tools

The miRanda algorithm (Griffiths-Jones et al., 2006) was used to scanfor potential miR-21 binding sites in 3′ UTR sequences of the human andmurine genome. Subsequently, Karlin-Altschul normalization was performed(miRBase Targets version 4.0; http://microrna.sanger.ac.uk/targets/v4/).In detail, the inventors used the miRBase data base (Version 4, SangerInstitute; USA) and sorted potential miR-21 targets by using thealgorithms “high number of conserved species; >6”), “low p-value;<0.001” and high miRBase score; >15. This procedure revealed 22 knowngenes to be potential miR-21 targets, of which 8 were previously shownto be expressed within heart tissue based on their GEO-expressionprofile (http://www.ncbi.nlm.nih.gov/geo/) (see Table 1). 5 genes wereadditionally predicted as miR-21 targets using the PicTar miRNA database 9 (Krek et al., 2005) (http://pictar.bio.nyu.edu/). Using theTargetScan miRNA target prediction software (Whitehead Institute forBiomedical Research, USA, Release 4.0; July 2007;http://www.targetscan.org/) the inventors identified targets withconserved sites with 8-mer seed matches for miR-21 only for two targets.Spry1 was then studied in more detail.

Western Blotting

Protein lysates from explanted hearts or culturedfibroblasts/cardiomyocytes were prepared as described (Buitrago et al.,2005). Extracts (20-50 μg protein per lane) were mixed with sampleloading buffer and under reducing conditions separated on 10%SDS-polyacrylamide gels. Proteins were electrotransferred onto PVDFmembrane (Immun-Blot®, Bio-Rad). The bands were detected using achemiluminescence assay (ECL Plus, Amersham). The inventors used primaryantibodies against sprouty-1 (Santa Cruz, sc-30048, dilution1:250-1:500), Erk1/2 (Cell Signaling, #9102, dilution 1:1000);phospho-Erk1/2 (Cell Signaling, #9101, dilution 1:1000) and G-β (SantaCruz Biotechnology, sc-378, dilution 1:1000), as well as appropriatesecondary antibodies (Anti-mouse-HRP (Cell Signaling, #7076, dilution1:10000; Anti-rabbit-HRP (Cell Signaling, #7074, dilution 1:10000).

In Vivo TAC Model and Modified Oligonucleotide Administration

The inventors used male C57BL/6 mice (10-12 weeks old, 25 g) fromCharles River Laboratories (Sulzfeld, Germany). Transaortic constriction(TAC) was created using a 7-0 suture tied twice around the aorta and a27-gauge needle. The needle was then gently retracted, yielding an about80% constriction of the aorta. During the same operations, a jugularvein catheter was implanted by standard surgical procedures. Thesequences were: antagomir-21,5′-oUsoCsoAoAoCoAoUoCoAoGoUoCoUoGoUoAoAoGsoCsoUsoAs-Chol-3′. Allnucleotides used in synthesis are 2′-OMe-modified (Subscript ‘o’).Subscript ‘s’ represents a phosphorothioate linkage; “Cy3” indicates Cy3dye label at the 5′ end of the oligo; “Chol” represents cholesterollinked through a hydroxyprolinol linkage. Treatment started 24 h postTAC and animals received PBS or miR-21 antagonist injections via animplanted jugular vein catheter (three consecutive days, jugular veininjections of PBS or miR-21 antagonist at doses of 80 mg per kg bodyweight in 0.2 ml per injection. As a positive control for effectivecardiac delivery Cy-3-labeled modified oligonucleotide (antagomir-181a)(80 mg/kg) was injected into the jugular vein catheter and the heart wasremoved 3 h later, fixed and Cy3 staining observed by fluorescencemicroscopy.

Cardiac Functional Analysis

After 3 weeks of TAC, mice were anesthetized with isoflurane, andcardiac dimensions and function were analyzed by 15-MHz pulse-waveDoppler echocardiography. Then the heart was removed, weighted afterremoval of atria and subjected to further analysis. Echocardiographicstudies were performed under light anesthesia with spontaneousrespiration using isoflurane. Two independent ultrasonographerexperienced in rodent imaging and blinded to the experimental groupsperformed the echocardiography, operating a Toshiba Power Vision 6000with a 15 MHz transducer. 2D left-parasternal short-axis views at thelevel of the papillary muscles were recorded. Correct probe placementwas judged by the round appearance of the left ventricular (LV) cavityafter angulation and craniocaudal transducer movements. LV end-diastolicarea was calculated by manual tracings of the endocardial borderfollowed by planimetry with the Nice software package (Toshiba MedicalSystems). Simultaneous transversal M-mode tracings were recorded withthe cursor placed in the middle of the LV cavity. Fractional shorteningwas calculated as described (Collins et al., 2001).

Detection of Cardiac Fibrosis

Mouse hearts were fixed in 4% buffered formalin and embedded inparaffin. 5 μm picrosirius red sections were examined using a NikonECLIPSE 50i microscope equipped with filters to provide circularlypolarized illumination (Whittaker et al., 1994). The lower filter wasplaced above the microscope's field iris diaphragm ring, while the upperfilter was constructed from a combination of a quarter-wave plate placedbelow a linear polarizer aligned such that its transmission axis was at45° to the fast axis of the wave plate. These two filters were crossed,that is aligned so that the background in the field of view was as darkas possible. Tissue images were obtained with a 20× objective lens,recorded on a cooled digital camera (DS-5Mc, Nikon), and analyzed usingSigmaScan Pro 5.0 image analysis software (SPSS Inc.,USA). Precisely,original (circularly polarized) images, were resolved each into itscyan, yellow, magenta and black components (using the automated functionCYMK provided by the image-analysis). The black component was subtractedfrom the polarized image. Prior to subtraction the black component wasadequately brightness adjusted to ensure the elimination of theinterstitial space and non-collagen elements but not of the thinnestcollagen fibers (confirmed by inspection). Then the subtracted image wassubjected to a final color separation into its hue, saturation, value(HSV) components using the automated function HSV provided by the imagesoftware. A histogram of hue frequency was obtained from the resolved8-bit hue image, which contains 256 colors. The following huedefinitions were used: red 2-9 and 230-256, orange 10-38, yellow 39-51,green 52-128. The hue range 129-229 consists of interstitial space andnon-birefringent tissue elements. The number of pixels within hue red,orange, yellow and green ranges was determined, and expressed as apercentage of the total number of collagen pixels, which in turn wasexpressed as a percentage of the total number of pixels in the image.

Fibroblast Apoptosis and FGF-2 Production

Cardiac fibroblasts obtained by the preplating step during cardiomyocyteisolation were cultured until sub-confluence. Cell purity was >95% basedon staining with the fibroblast marker anti-rat prolyl-4-hydroxylase(Acris Antibodies, AF5110-1; data not shown). After treatment withmiR-21 precursors, inhibitors or respective controls (see above),Annexin V-positive fibroblasts were measured by FACS analysis(Annexin-VFLUOS kit, Roche Diagnostics GmbH, Mannheim, Germany).Enzyme-linked immunosorbent assay (ELISA) was performed to quantifyFGF-2 concentrations in supernatants of miR-21 modulated cardiacfibroblasts. FGF-2-determination was carried out using the QuantikineFGF Basic Immunoassay kit (R&D Systems, Minneapolis, USA) according tothe manufacturer's specifications.

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1.-139. (canceled)
 140. A method for diagnosing fibrosis comprising measuring the expression of miR-21 in a sample from a patient supposed to suffer from fibrosis, wherein an elevated level of miR-21 in comparison to a control sample indicates fibrosis or a predisposition thereof.
 141. The method of claim 140 comprising contacting the sample with a modified oligonucleotide complementary to miR-21 (SEQ ID NO: 1).
 142. The method of claim 141, wherein each of a plurality of nucleosides of the modified oligonucleotide comprises a modified sugar.
 143. The method of claim 142, wherein each modified sugar is independently selected from a 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or a bicyclic sugar moiety.
 144. The method of claim 141, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.
 145. The method of claim 141, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
 146. The method of claim 141, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
 147. The method of claim 141, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
 148. A method for screening a pharmaceutically active compound for the treatment and/or the prevention of fibrosis or a predisposition thereof, comprising a) contacting a candidate substance with a sample comprising miR-21; and b) determining the effect of the candidate substance on the sample; wherein an alteration of miR-21 indicates that the candidate substance is a pharmaceutically active compound.
 149. The method of claim 148, wherein the candidate substance is a modified oligonucleotide complementary to miR-21 (SEQ ID NO: 1).
 150. The method of claim 149, wherein each of a plurality of nucleosides of the modified oligonucleotide comprises a modified sugar.
 151. The method of claim 150, wherein each modified sugar is independently selected from a 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or a bicyclic sugar moiety.
 152. The method of claim 149, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.
 153. The method of claim 149, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
 154. The method of claim 149, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
 155. The method of claim 149, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1). 